Combination therapy of T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists

ABSTRACT

The present invention generally relates to T cell activating bispecific antigen binding molecules, PD-1 axis binding antagonists, and in particular to combination therapies employing such T cell activating bispecific antigen binding molecules and PD-1 axis binding antagonists, and their use of these combination therapies for the treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/600,011, filed May 19, 2017, which is a continuation of InternationalApplication No. PCT/EP2015/076682, filed Nov. 16, 2015, which claimspriority to European Patent Application No. 15167173.2, filed May 5,2015, European Patent Application No. 15152141.6, filed Jan. 22, 2015,and European Patent Application No. 14194136.9, filed Nov. 20, 2014.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 18, 2017, isnamed 51177-012002_Sequence_Listing_8.6.20_ST25.txt and is 527, 213bytes in size.

FIELD OF THE INVENTION

The present invention relates to combination therapies employing T cellactivating bispecific antigen binding molecule and a PD-1 axis bindingantagonist, and, optionally, a TIM3 antagonist, and the use of thesecombination therapies for the treatment of cancer.

BACKGROUND

Monoclonal antibodies are powerful therapeutic agents for the treatmentof cancer that selectively target antigens which are differentiallyexpressed on cancer cells.

Bispecific antibodies designed to bind with one antigen binding moietyto a surface antigen on target cells, and with the second antigenbinding moiety to an activating, invariant component of the T cellreceptor (TCR) complex, have become of interest in recent years. Thesimultaneous binding of such an antibody to both of its targets willforce a temporary interaction between target cell and T cell, causingactivation of any cytotoxic T cell and subsequent lysis of the targetcell. Hence, the immune response is re-directed to the target cells andis independent of peptide antigen presentation by the target cell or thespecificity of the T cell as would be relevant for normal MHC-restrictedactivation of CTLs. In this context it is crucial that CTLs are onlyactivated when a target cell is presenting the bispecific antibody tothem, i.e., the immunological synapse is mimicked. Particularlydesirable are bispecific antibodies that do not require lymphocytepreconditioning or co-stimulation in order to elicit efficient lysis oftarget cells. It is not well understood how TCBs affect the T cellitself beyond activation of certain effector function.

Activation of resting T lymphocytes, or T cells, by antigen-presentingcells (APCs) appears to require two signal inputs. Lafferty et al, Aust.J. Exp. Biol. Med. ScL 53: 27-42 (1975). The primary, or antigenspecific, signal is transduced through the T-cell receptor (TCR)following recognition of foreign antigen peptide presented in thecontext of the major histocompatibility-complex (MHC). The second, orco-stimulatory, signal is delivered to T-cells by co-stimulatorymolecules expressed on antigen-presenting cells (APCs), and promotesT-cell clonal expansion, cytokine secretion and effector function.Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). In the absence ofco-stimulation, T cells can become refractory to antigen stimulation, donot mount an effective immune response, and may result in exhaustion ortolerance to foreign antigens.

T cells can receive both positive and negative secondary co-stimulatorysignals. The balance of positive and negative signals is important toelicit effective immune responses, while maintaining immune toleranceand preventing autoimmunity. Negative secondary signals appear necessaryfor induction of T-cell tolerance, while positive signals promote T cellactivation.

Recently, it has been discovered that T cell dysfunction or anergyoccurs concurrently with an induced and sustained expression of theinhibitory receptor, programmed death 1 polypeptide (PD-1). One of itsligands, PD-L1 is overexpressed in many cancers and is often associatedwith poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813)(Thompson R H et al., Cancer Res 2006, 66(7):3381). Interestingly, themajority of tumor infiltrating T lymphocytes predominantly express PD-1,in contrast to T lymphocytes in normal tissues and peripheral blood Tlymphocytes indicating that up-regulation of PD-1 on tumor-reactive Tcells can contribute to impaired antitumor immune responses (Blood 20091 14(8): 1537).

T cell Immunoglobulin- and Mucin domain-containing molecule 3 (TIM3), isimportant in immune regulation. This cell surface protein is expressed,preferentially, by type 1 T helper cells and has been implicated in theregulation of macrophage activation, inflammatory conditions and cancer(Majeti R et al., PNAS, 106 (2009) 3396-3401 and WO2009/091547). Bindingof TIM-3 to one of its ligands (e.g., galectin-9) can suppress the Th1response by inducing programmed cell death, thereby supportingperipheral tolerance. Treatment with TIM-3 siRNA or with an anti-TIM-3antagonist antibody increases secretion of interferon alpha from CD4positive T-cells, supporting the inhibitory role of TIM-3 in human Tcells. Examples of the anti-TIM-3 monoclonal antibodies include aredisclosed in WO2013/06490 and US2012/189617 (Ngiow et al., Cancer Res7:6567 (2011)).

FOLR1 is expressed on tumor cells of various origins, e.g., ovarian andlung cancer. Several approaches to target FOLR1 with therapeuticantibodies, such as farletuzumab, antibody drug conjugates, or adoptiveT cell therapy for imaging of tumors have been described (Kandalaft etal., J Transl Med. 2012 Aug. 3; 10:157. doi: 10.1186/1479-5876-10-157;van Dam et al., Nat Med. 2011 Sep. 18; 17(10):1315-9. doi:10.1038/nm.2472; Clifton et al., Hum Vaccin. 2011 February; 7(2):183-90.Epub 2011 Feb. 1; Kelemen et al., Int J Cancer. 2006 Jul. 15;119(2):243-50; Vaitilingam et al., J Nucl Med. 2012 July; 53(7); Teng etal., 2012 August; 9(8):901-8. doi: 10.1517/17425247.2012.694863. Epub2012 Jun. 5. Some attempts have been made to target folatereceptor-positive tumors with constructs that target the folate receptorand CD3 (Kranz et al., Proc Natl Acad Sci USA. Sep. 26, 1995; 92(20):9057-9061; Roy et al., Adv Drug Deliv Rev. 2004 Apr. 29; 56(8):1219-31;Huiting Cui et al Biol Chem. Aug. 17, 2012; 287(34): 28206-28214; Lamerset al., Int. J. Cancer. 60(4):450 (1995); Thompson et al., MAbs. 2009July-August; 1(4):348-56. Epub 2009 Jul. 19; Mezzanzanca et al., Int. J.Cancer, 41, 609-615 (1988).

There remains a need for such an optimal therapy for treating,stabilizing, preventing, and/or delaying development of various cancers.

SUMMARY

Broadly, the present invention relates to bispecific antibodiescombining a Folate Receptor 1 (FolR1) targeting antigen binding sitewith a second antigen binding site that targets CD3 and their use incombination with a PD-1 axis binding antagonist, e.g., for the treatmentof cancer. In one embodiment, the combination further comprises a TIM3antagonist. The methods and combinations of the present invention enableenhanced immunotherapy. The advantage over conventional treatment is thespecificity of inducing T cell activation only at the site where FolR1is expressed as well as the reduction and/or reversal of low T cellmediated activity also termed T cell exhaustion due to the combinationwith a PD-1 axis binding antagonist, and, optionally, a TIM3 antagonist.

Accordingly, in one aspect, the present invention provides a method fortreating or delaying progression of a cancer in an individual comprisingadministering to the individual an effective amount of a T cellactivating bispecific antigen binding molecule and a PD-1 axis bindingantagonist. In one embodiment, the T cell activating bispecific antigenbinding molecule comprises a first antigen binding moiety capable ofspecific binding to CD3 and a second antigen binding moiety capable ofspecific binding to Folate Receptor 1 (FolR1). In one embodiment, thefirst antigen binding moiety comprises at least one heavy chaincomplementarity determining region (CDR) amino acid sequence selectedfrom the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:39 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34. In one embodiment, the firstantigen binding moiety comprises a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31. In one embodiment,the T cell activating bispecific antigen binding molecule furthercomprises a third antigen binding moiety capable of specific binding toFolR1. In one embodiment, the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one embodiment, the third antigen binding moiety is identical to thesecond antigen binding moiety. In one embodiment, at least one of thefirst, second and third antigen binding moiety is a Fab molecule.

In one embodiment, the antigen binding moiety capable of specificbinding to Folate Receptor 1 (FolR1) comprises at least one heavy chaincomplementarity determining region (CDR) amino acid sequence selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34. In one embodiment, the antigenbinding moiety capable of specific binding to Folate Receptor 1 (FolR1)comprises a variable heavy chain comprising an amino acid sequence ofSEQ ID NO: 15 and a variable light chain comprising an amino acidsequence of SEQ ID NO: 31. In one embodiment, the antigen binding moietycapable of specific binding to Folate Receptor 1 (FolR1) comprises atleast one heavy chain complementarity determining region (CDR) aminoacid sequence selected from the group consisting of SEQ ID NO: 8, SEQ IDNO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected fromthe group of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 65. In oneembodiment, the antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprises a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 55 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 64. In one embodiment,the antigen binding moiety capable of specific binding to FolateReceptor 1 (FolR1) comprises at least one heavy chain complementaritydetermining region (CDR) amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at leastone light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54. In one embodiment, the antigen binding moiety capableof specific binding to FolR1 comprises:

-   -   a) a complementarity determining region heavy chain 1 (CDR-H1)        amino acid sequences of SEQ ID NO: 8;    -   (b) a CDR-H2 amino acid sequence of SEQ ID NO: 9;    -   (c) a CDR-H3 amino acid sequence of SEQ ID NO: 50;    -   (d) a complementarity determining region light chain 1 (CDR-L1)        amino acid sequence of SEQ ID NO: 52;    -   (e) a CDR-L2 amino acid sequence of SEQ ID NO: 53, and    -   (f) a CDR-L3 amino acid sequence of SEQ ID NO: 54.

In one such embodiment, the antigen binding moiety capable of specificbinding to FolR1 comprises a variable heavy chain comprising an aminoacid sequence of SEQ ID NO: 49 and a variable light chain comprising anamino acid sequence of SEQ ID NO: 51.

In one embodiment, the T cell activating bispecific antigen bindingmolecule binds to a human FolR1, a cynomolgus monkey FolR1 and a murineFolR1.

In one embodiment, the T cell activating bispecific antigen bindingmolecule induces proliferation of a human CD3 positive T cell in vitro.

In one embodiment, the T cell activating bispecific antigen bindingmolecule induces human peripheral blood mononuclear cell mediatedkilling of a FolR1-expressing human tumor cell in vitro.

In one embodiment, the T cell activating bispecific antigen bindingmolecule induces T cell mediated killing of a FolR1-expressing humantumor cell in vitro. In one embodiment, the T cell activating bispecificantigen binding molecule induces T cell mediated killing of theFolR1-expressing human tumor cell in vitro with an EC50 of between about36 pM and about 39573 pM after 24 hours. In one embodiment, the T cellactivating bispecific antigen binding molecule induces upregulation ofcell surface expression of at least one of CD25 and CD69 on the T cellas measured by flow cytometry. In one embodiment, the T cell activatingbispecific antigen binding molecule binds human FolR1 with an apparentK_(D) of about 5.36 pM to about 4 nM. In one embodiment, the T cellactivating bispecific antigen binding molecule binds human andcynomolgus FolR1 with an apparent K_(D) of about 4 nM. In oneembodiment, the T cell activating bispecific antigen binding moleculebinds murine FolR1 with an apparent K_(D) of about 1.5 nM. In oneembodiment, the T cell activating bispecific antigen binding moleculebinds human FolR1 with a monovalent binding K_(D) of at least about 1000nM. In one embodiment, the T cell activating bispecific antigen bindingmolecule binds to FolR1 expressed on a human tumor cell. In oneembodiment, the T cell activating bispecific antigen binding moleculebinds to a conformational epitope on human FolR1. In one embodiment, theT cell activating bispecific antigen binding molecule does not bind tohuman Folate Receptor 2 (FolR2) or to human Folate Receptor 3 (FolR3).In one embodiment, the antigen binding moiety binds to a FolR1polypeptide comprising the amino acids 25 to 234 of human FolR1 (SEQ IDNO:227). In one embodiment, the FolR1 antigen binding moiety binds to aFolR1 polypeptide comprising the amino acid sequence of SEQ ID NOs:227,230 and 231, and wherein the FolR1 antigen binding moiety does not bindto a FolR polypeptide comprising the amino acid sequence of SEQ IDNOs:228 and 229. In one embodiment, the T cell activating bispecificantigen binding molecule comprises a) a first antigen-binding site thatcompetes for binding to human FolR1 with a reference antibody comprisinga variable heavy chain domain (VH) of SEQ ID NO: 49 and a variable lightchain domain of SEQ ID NO: 51; and b) a second antigen-binding site thatcompetes for binding to human CD3 with a reference antibody comprising avariable heavy chain domain (VH) of SEQ ID NO: 36 and a variable lightchain domain of SEQ ID NO: 31, wherein binding competition is measuredusing a surface plasmon resonance assay.

In one embodiment, the T cell activating bispecific antigen bindingmolecule comprises a first, a second, a third, a fourth and a fifthpolypeptide chain that form a first, a second and a third antigenbinding moiety, wherein the first antigen binding moiety is capable ofbinding CD3 and the second and the third antigen binding moiety each arecapable of binding Folate Receptor 1 (FolR1), wherein a) the first andthe second polypeptide chain comprise, in amino (N)-terminal to carboxyl(C)-terminal direction, VLD1 and CLD1; b) the third polypeptide chaincomprises, in N-terminal to C-terminal direction, VLD2 and CH1D2; c) thefourth polypeptide chain comprises, in N-terminal to C-terminaldirection, VHD1, CH1D1, CH2D1 and CH3D1; d) the fifth polypeptide chaincomprises VHD1, CH1D1, VHD2, CLD2, CH2D2 and CH3D2; wherein

-   -   VLD1 is a first light chain variable domain    -   VLD2 is a second light chain variable domain    -   CLD1 is a first light chain constant domain    -   CLD2 is a second light chain constant domain    -   VHD1 is a first heavy chain variable domain    -   VHD2 is a second heavy chain variable domain    -   CH1D1 is a first heavy chain constant domain 1    -   CH1D2 is a second heavy chain constant domain 1    -   CH2D1 is a first heavy chain constant domain 2    -   CH2D2 is a second heavy chain constant domain 2    -   CH3D1 is a first heavy chain constant domain 3    -   CH3D2 is a second heavy chain constant domain 3.

In one such embodiment,

-   -   a. the third polypeptide chain and VHD2 and CLD2 of the fifth        polypeptide chain form the first antigen binding moiety capable        of binding CD3;    -   b. the first polypeptide chain and VHD1 and CH1D1 of the fourth        polypeptide chain form the second binding moiety capable of        binding to FolR1; and    -   c. the second polypeptide chain and VHD1 and CH1D1 of the fifth        polypeptide chain form the third binding moiety capable of        binding to FolR1.

In one such embodiment, the first and second polypeptide chain comprisethe amino acid sequence of SEQ ID NO:399. In one such embodiment, thethird polypeptide chain comprises the amino acid sequence of SEQ IDNO:86. In one such embodiment, the fourth polypeptide chain comprisesthe amino acid sequence of SEQ ID NO:394. In one such embodiment, thefifth polypeptide chain comprises the amino acid sequence of SEQ IDNO:397. In one embodiment,

-   -   a. the first and second polypeptide chain comprise the amino        acid sequence of SEQ ID NO:399;    -   b. the third polypeptide chain comprises the amino acid sequence        of SEQ ID NO:86;    -   c. the fourth polypeptide chain comprises the amino acid        sequence of SEQ ID NO:394; and    -   d. the fifth polypeptide chain comprise the amino acid sequence        of SEQ ID NO:397.

In some embodiments, the bispecific antibody is bivalent both for FolR1and CD3.

In some embodiments, the bispecific antibody comprises one or more Fabfragment(s) comprising an antigen binding site specific for CD3, whereinthe variable regions or the constant regions of the heavy and lightchain are exchanged.

In some embodiments, the bispecific antibody comprises an Fc domain, atleast one Fab fragment comprising the antigen binding site specific forFolR1, and at least one Fab fragment comprising the antigen binding sitespecific for CD3 wherein either the variable regions or the constantregions of the heavy and light chain of at least one Fab fragment areexchanged.

In some embodiments, the bispecific antibody comprises:

-   -   a) an Fc domain,    -   b) a first and second Fab fragment each comprising an antigen        binding site specific for FolR1,    -   c) a third Fab fragment comprising an antigen binding site        specific for CD3, wherein the third Fab fragment is connected at        the C-terminus of the variable heavy chain (VH) to the second        subunit of the Fc domain and wherein the third Fab fragment is        connected at the N-terminus of the variable heavy chain to the        C-terminus of the second Fab fragment.

In one embodiment at least one of said Fab fragments is connected to theFc domain via a peptide linker.

In one embodiment said bispecific antibody comprises an Fc domain, whichcomprises one or more amino acid substitution that reduces binding to Fcreceptors and/or effector function. In one embodiment said one or moreamino acid substitution is at one or more positions selected from thegroup of L234, L235, and P329. In one embodiment each subunit of the Fcdomain comprises three amino acid substitutions that abolish binding toan activating or inhibitory Fc receptor and/or effector function whereinsaid amino acid substitutions are L234A, L235A and P329G.

In some embodiments, the PD-1 axis binding antagonist is selected fromthe group consisting of a PD-1 binding antagonist, a PDL1 bindingantagonist and a PDL2 binding antagonist.

In some embodiments, the PD-1 axis binding antagonist is a PD-bindingantagonist. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to its ligand binding partners. In some embodiments,the PD-1 binding antagonist inhibits the binding of PD-1 to PDL1. Insome embodiments, the PD-binding antagonist inhibits the binding of PD-1to PDL2. In some embodiments, the PD-1 binding antagonist inhibits thebinding of PD-1 to both PDL1 and PDL2. In some embodiments, PD-1 bindingantagonist is an antibody. In some embodiments, the anti-PD-1 antibodyis a monoclonal antibody. In some embodiments, the anti-PD-1 antibody isan antibody fragment selected from the group consisting of Fab, Fab′-SH,Fv, scFv, and (Fab′)2 fragments. In some embodiments, PD-1 bindingantagonist is nivolumab, pembrolizumab, CT-011, or AMP-224.

In some embodiments, the PD-1 axis binding antagonist is a PDL1 bindingantagonist. In some embodiments, the PDL1 binding antagonist inhibitsthe binding of PDL1 to PD-1. In some embodiments, the PDL1 bindingantagonist inhibits the binding of PDL1 to B7-1. In some embodiments,the PDL1 binding antagonist inhibits the binding of PDL1 to both PD-1and B7-1. In some embodiments, the PDL1 binding antagonist is ananti-PDL1 antibody. In some embodiments, the anti-PDL1 antibody is amonoclonal antibody. In some embodiments, the anti-PDL1 antibody is anantibody fragment selected from the group consisting of Fab, Fab′-SH,Fv, scFv, and (Fab′)2 fragments. In some embodiments, the anti-PDL1antibody is a humanized antibody or a human antibody. In someembodiments, the PDL1 binding antagonist is selected from the groupconsisting of: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736.

In some embodiments, the anti-PDL1 antibody comprises a heavy chaincomprising HVR-H1 sequence of SEQ ID NO:289, HVR-H2 sequence of SEQ IDNO:290, and HVR-H3 sequence of SEQ ID NO:291; and a light chaincomprising HVR-L1 sequence of SEQ ID NO:292, HVR-L2 sequence of SEQ IDNO:293, and HVR-L3 sequence of SEQ ID NO:294. In some embodiments,anti-PDL1 antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:280 or SEQ ID NO:281 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:383. In some embodiments, the anti-PDL1 antibody comprises a heavychain comprising the amino acid sequence of SEQ ID NO:278 and/or a lightchain comprising the amino acid sequence of SEQ ID NO:279.

In some embodiments, the PD-1 axis binding antagonist is a PDL2 bindingantagonist. In some embodiments, PDL2 binding antagonist is an antibody.In some embodiments, the anti-PDL2 antibody is a monoclonal antibody. Insome embodiments, the anti-PDL2 antibody is an antibody fragmentselected from the group consisting of Fab, Fab′-SH, Fv, scFv, and(Fab′)2 fragments. In some embodiments, PDL2 binding antagonist is animmunoadhesin.

In one embodiment, the method of any of the above embodiments furthercomprises administering to the individual a T cell immunoglobulin mucin3 (TIM3) antagonist. In one embodiment, the TIM3 antagonist is ananti-TIM3 antibody. In one embodiment, the anti-TIM3 antibody inducesinternalization of TIM3 on a TIM3 expressing cell of at least 45% after120 Minutes at 37° C. wherein internalization is measured by FACSanalysis. In one embodiment, the anti-TIM3 antibody has one or more ofthe following properties:

-   -   a) competes for binding to TIM3 with an anti-Tim3 antibody        comprising the VH of SEQ ID NO:7 and VL of SEQ ID NO: 8    -   b) binds to a human and cynomolgoues TIM3    -   c) shows as immunoconjugate a cytotoxic activity on TIM3        expressing cells    -   d) induces interferon-gamma release.

In one embodiment, the anti-TIM3 antibody has one or more of thefollowing properties:

-   -   a. competes for binding to TIM3 with an anti-Tim3 antibody        comprising the VH of SEQ ID NO:7 and VL of SEQ ID NO: 8    -   b. binds to a human and cynomolgoues TIM3    -   c. shows as immunoconjugate a cytotoxic activity on TIM3        expressing cells    -   d. induces interferon-gamma release.

In one embodiment, the anti-TIM3 antibody is a monoclonal antibody. Inone embodiment, the anti-TIM3 antibody is a human, humanized, orchimeric antibody. In one embodiment, the anti-TIM3 antibody is anantibody fragment that binds to TIM3. In one embodiment, the anti-TIM3antibody is Fab fragment. In one embodiment, the anti-TIM3 antibodycomprises:

-   -   A) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:304, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:305, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:306; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:307; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:308 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:309; or    -   B) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:304, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:305, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:306; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:314; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:308 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:309; or    -   C) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:304, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:305, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:306; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:315; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:308 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:309; or    -   D) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:316, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:317, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:318; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:319; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:320 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:321; or    -   E) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:324, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:325, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:326; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:327; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:328 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:329; or.    -   F) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:332, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:333, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:334; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:335; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:336 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:337; or    -   G) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:340, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:341, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:342; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:343; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:344 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:345; or    -   H) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:348, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:349, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:350; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:351; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:352 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:353; or    -   I) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:356, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:357, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:358; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:359; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:360 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:361; or    -   J) (a) a VH domain comprising (i) HVR-H1 comprising the amino        acid sequence of SEQ ID NO:364, (ii) HVR-H2 comprising the amino        acid sequence of SEQ ID NO:365, and (iii) HVR-H3 comprising an        amino acid sequence selected from SEQ ID NO:366; and (b) a VL        domain comprising (i) HVR-L1 comprising the amino acid sequence        of SEQ ID NO:367; (ii) HVR-L2 comprising the amino acid sequence        of SEQ ID NO:368 and (iii) HVR-L3 comprising the amino acid        sequence of SEQ ID NO:369.

In one embodiment, the anti-TIM3 antibody is a full length IgG1 antibodywith mutations S228P, L235E and P329G according to the EU index of Kabatnumbering. In one embodiment, the anti-TIM3 antibody is any one of theantibodies described in WO 2011/155607, WO 2013/006490, WO 03/063792, WO2009/097394, and WO 2011/159877. In one embodiment, the anti-TIM3antibody is F38-2E2.

In one embodiment, the cancer contains a KRAS wildtype. In oneembodiment, the cancer contains an activating KRAS mutation.

In one embodiment, the treatment results in a sustained response in theindividual after cessation of the treatment. In one embodiment, at leastone of the T cell activating bispecific antigen binding molecule and thePD-1 axis binding antagonist is administered continuously. In oneembodiment, at least one of the T cell activating bispecific antigenbinding molecule and the PD-1 axis binding antagonist is administeredintermittently. In one embodiment, the PD-1 axis binding antagonist isadministered before the FolR1 TCB. In one embodiment, the PD-1 axisbinding antagonist is administered simultaneous with the FolR1 TCB. Inone embodiment, the PD-1 axis binding antagonist is administered afterthe FolR1 TCB. In one embodiment, the cancer is selected from the groupconsisting of ovarian cancer, lung cancer, breast cancer, renal cancer,colorectal cancer, endometrial cancer. In one embodiment, at least oneof the T cell activating bispecific antigen binding molecule and thePD-1 axis binding antagonist is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally.

In one embodiment, T cells in the individual have enhanced activation,proliferation and/or effector function relative to prior to theadministration of the combination. In one embodiment, T cells in theindividual have enhanced activation, proliferation and/or effectorfunction relative to administration of the T cell activating bispecificantigen binding molecule alone. In one embodiment, T cell effectorfunction is secretion of at least one of IL-2, IFN-γ and TNF-α. In oneembodiment, the individual comprises less than about 15% PD-1^(hi)expressing tumor-infiltrating T cells.

In one aspect, the invention provides for a method of enhancing immunefunction in an individual having a FolR1 positive cancer comprisingadministering to the individual an effective amount of a combination ofa T cell activating bispecific antigen binding molecule specific forFolate Receptor 1 (FolR1) and CD3, and a PD-1 axis binding antagonist.In one embodiment, T cells in the individual have enhanced activation,proliferation and/or effector function relative to prior to theadministration of the combination. In one embodiment, T cells in theindividual have enhanced activation, proliferation and/or effectorfunction relative to administration of the T cell activating bispecificantigen binding molecule alone. In one embodiment, T cell effectorfunction is secretion of at least one of IL-2, IFN-γ and TNF-α.

In one embodiment, the individual comprises less than about 15%PD-1^(hi) expressing tumor-infiltrating T cells.

In another aspect, the invention provides for a method for selecting apatient for treatment with a combination of a T cell activatingbispecific antigen binding molecule specific for Folate Receptor 1(FolR1) and CD3, and a PD-1 axis binding antagonist comprising measuringthe level of PD-1 expression, wherein a patient having less than about15% PD-1^(hi) expressing T cells is selected for treatment with thecombination.

In another aspect, the invention provides for a kit comprising a T cellactivating bispecific antigen binding molecule specific for FolateReceptor 1 (FolR1) and CD3, and a package insert comprising instructionsfor using the T cell activating bispecific antigen binding molecule witha PD-1 axis binding antagonist to treat or delay progression of cancerin an individual. In one embodiment, the kit further comprisesinstructions for using the T cell activating bispecific antigen bindingmolecule with a TIM3 antagonist.

In another aspect, the invention provides for a kit comprising a T cellactivating bispecific antigen binding molecule specific for FolateReceptor 1 (FolR1) and CD3 and a PD-1 axis binding antagonist, and apackage insert comprising instructions for using the T cell activatingbispecific antigen binding molecule and the PD-1 axis binding antagonistto treat or delay progression of cancer in an individual. In oneembodiment, the kit further comprises a TIM3 antagonist. In oneembodiment, the PD-1 axis binding antagonist is an anti-PD-1 antibody oran anti-PDL-1 antibody. In one embodiment, the PD-1 axis bindingantagonist is an anti-PD-1 immunoadhesin.

In another aspect, the invention provides for a pharmaceuticalcomposition comprising a T cell activating bispecific antigen bindingmolecule specific for Folate Receptor 1 (FolR1) and CD3, a PD-1 axisbinding antagonist and a pharmaceutically acceptable carrier. In oneembodiment, the pharmaceutical composition further comprises a TIM3antagonist.

In another aspect, the invention provides for a use of a combination ofa T cell activating bispecific antigen binding molecule specific forFolate Receptor 1 (FolR1) and CD3 and a PD-1 axis binding antagonist inthe manufacture of a medicament for the treatment of cancer. In oneembodiment, the medicament is for treatment of ovarian cancer, lungcancer, breast cancer, renal cancer, colorectal cancer, endometrialcancer.

In certain embodiments of all aspects of the present invention,advantageously said T cell activating bispecific antigen bindingmolecule and/or PD-1 axis binding antagonist is human or humanized.

In some embodiments, the bispecific antibody comprises an Fc domain, atleast one Fab fragment comprising the antigen binding site specific forFolR1, and at least one Fab fragment comprising the antigen binding sitespecific for CD3.

In one aspect, the invention provides for a method for treating ordelaying progression of a cancer in an individual comprisingadministering to the individual an effective amount of a T cellactivating bispecific antigen binding molecule and a TIM3 antagonist. Insome embodiments, the T cell activating bispecific antigen bindingmolecule comprises an Fc domain, two Fab fragments comprising each anantigen binding site specific for FolR1, and one Fab fragment comprisingan antigen binding site specific for CD3.

In a further aspect, the present invention provides the use of acombination of a T cell activating bispecific antigen binding moleculethat binds to FolR1 and CD3, and a PD-1 axis binding antagonist in themanufacture of a medicament for the treatment of cancer.

In a further aspect, the present invention provides the use of acombination of a T cell activating bispecific antigen binding moleculethat binds to FolR1 and CD3, a PD-1 axis binding antagonist and a TIM3antagonist in the manufacture of a medicament for the treatment ofcancer.

Embodiments of the present invention will now be described by way ofexample and not limitation with reference to the accompanying figures.However various further aspects and embodiments of the present inventionwill be apparent to those skilled in the art in view of the presentdisclosure.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-I illustrate exemplary configurations of the T cell activatingbispecific antigen binding molecules (TCBs) of the invention. Allconstructs except the kappa-lambda format in (FIG. 1I) have P329G LALAmutations and comprise knob-into-hole Fc fragments with knob-into-holemodifications. (FIG. 1A) Illustration of the “FolR1 TCB 2+1 inverted(common light chain)”. The FolR1 binder is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first subunit of the Fcdomain comprising the knob modification. These constructs are notcrossed and have three times the same VLCL light chain. (FIG. 1B)Illustration of the “FolR1 TCB 1+1 head-to-tail (common light chain)”.These constructs are not crossed and have two times the same VLCL lightchain. (FIG. 1C) Illustration of the “FolR1 TCB 1+1 classical (commonlight chain)”. These constructs are not crossed and have two times thesame VLCL light chain. (FIG. 1D) Illustration of the “FolR1 TCB 2+1classical (common light chain)”. The CD3 binder is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first subunitof the Fc domain comprising the knob modification. These constructs arenot crossed and have three times the same VLCL light chain. (FIG. 1E)Illustration of the “FolR1 TCB 2+1 crossfab classical”. These constructscomprise a Ck-VH chain for the CD3 binder instead of the conventionalCH1-VH chain. The CD3 binder is fused at the C-terminus of the Fab heavychain to the N-terminus of the first subunit of the Fc domain comprisingthe knob modification. (FIG. 1F) Illustration of the “FolR1 TCB 2+1crossfab inverted”. These constructs comprise a Ck-VH chain for the CD3binder instead of the conventional CH1-VH chain. The FolR1 binder isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst subunit of the Fc domain comprising the knob modification. (FIG.1G) Illustration of the “FolR1 TCB 1+1 crossfab head-to-tail”. Theseconstructs comprise a Ck-VH chain for the CD3 binder instead of theconventional CH1-VH chain. (FIG. 1H) Illustration of the “FolR1 TCB 1+1crossfab classical”. These constructs comprise a Ck-VH chain for the CD3binder instead of the conventional CH1-VH chain. FIG. 1I illustrates theCD3/FolR1 kappa-lambda antibody format. These constructs comprise acrossed common light chain VLCH1 and one crossed VHCL chain specific forCD3 and one crossed VHCL chain specific for FolR1.

FIGS. 2A-C depict graphs summarizing Binding of FoLR1 IgG binders toHeLa cells. Binding of newly generated FolR1 binders to FolR1 expressedon HeLa cells were determined by flow cytometry. Bound antibodies weredetected with a fluorescently labeled anti-human secondary antibody.

FIGS. 3A-B depict graphs summarizing specificity of FolR1 binders forFolR1. Binding of FolR1 IgGs to HEK cells transiently transfected witheither FolR1 or FolR2 was analyzed by flow cytometry to identify cloneswhich bind specifically to FolR1 and not to FolR2. The antibodies weredetected with a fluorescently labeled anti-human secondary antibody.

FIGS. 4A-B depict graphs summarizing cross-reactivity of FolR1 bindersto cyFoLR1. Cross-reactivity of the FolR1 antibodies to cyno FolR1 wasaddressed on HEK cells transiently transfected with cyFolR1 by flowcytometry. The antibodies were detected with a fluorescently labeledanti-human secondary antibody.

FIG. 5 depicts a graph illustrating internalization of FolR1 TCBs afterbinding. Internalization of the four FolR1 TCBs after binding to FolR1was tested on HeLa cells. Remaining FolR1 TCBs on the surface weredetected with a fluorescently labeled anti-human secondary antibodyafter indicated time points of incubation at 37° C. Percentage ofinternalization was calculated.

FIGS. 6A-E depict graphs summarizing binding of FolR1 IgGs to cells withdifferent FolR1 expression levels. Binding of 9D11, 16D5 and Mov19 IgGto tumor cells with different FolR1 expression levels was analyzed byflow cytometry. DP47 IgG was included as isotype control and MKN-45 wereincluded as FolR1 negative cell line. The antibodies were detected witha fluorescently labeled anti-human secondary antibody.

FIGS. 7A-L depict graphs summarizing T cell mediated killing of HT-29and SKOV3 cells. FolR1 TCBs were used to test T cell mediated killing ofHT-29 and SKOV3 tumor cells and upregulation of activation marker on Tcells upon killing. (FIGS. 7A-D) T cell mediated killing of HT-29 andSKOV3 cells in the presence of 9D11 FolR1 TCB and 16D5 FolR1 TCB wasmeasured by LDH release after 24 h and 48 h. DP47 TCB was included asnegative control. After 48 h incubation upregulation of the activationmarker CD25 and CD69 on CD8 T cells and CD4 T cells upon killing ofSKOV3 (FIGS. 7E-H) or HT-29 (FIG. 7I-L) tumor cells was assessed by flowcytometry.

FIG. 8 depicts a graph showing absence of anti-FolR1 binding toerythrocytes. Erythrocytes were gated as CD235a positive population andbinding of 9D11 IgG, 16D5 IgG, Mov19 IgG and DP47 IgG to this populationwas determined by flow cytometry. The antibodies were detected with afluorescently labeled anti-human secondary antibody.

FIGS. 9A-D depict graphs summarizing activation marker upregulation inwhole blood. CD25 and CD69 activation marker upregulation of CD4 T cellsand CD8 T cells 24 h after addition of 9D11 FolR1 TCB, 16D5 FolR1 TCB,Mov19 FolR1 TCB and DP47 TCB was analyzed by flow cytometry.

FIGS. 10A-C depict T-cell killing induced by 36F2 TCB, 16D5 TCB, 16D5TCB classical, 16D5 TCB 1+1 and 16D5 TCB HT of Hela (high FolR1) (FIG.24A), Skov-3 (medium FolR1) (FIG. 24B) and HT-29 (low FolR1) (FIG. 24C)human tumor cells (E:T=10:1, effectors human PBMCs, incubation time 24h). DP47 TCB was included as non-binding control.

FIGS. 11A-B show expression of inhibitory receptors ontumor-infiltrating T cells. CD8⁺ and CD4⁺ T cells in tumor samples werecharacterized by flow cytometry for their expression of inhibitoryreceptors.

FIGS. 12A-O show activation of CD8⁺ T cells in tumor digests andmalignant effusions upon exposure to FolR1-TCB. Tumor digests ormalignant effusions were cultured for 24 h in the presence or absence ofFolR1-TCB or the control TCB DP-47. The expression of activation markersor markers of T cell function on CD8⁺ T cells was determined by flowcytometry (FIG. 12A-M). FIG. 12J-K show representative FACS plotsshowing FolR1-TCB-induced T cell activation in a high responding(BS-269) or a low responding patient (BS-212). FIG. 12L depicts FACSplots showing FolR1-TCB-induced activation marker expression in T cellsfrom a representative patient. The graphs in FIG. 12M depict theincrease in marker expression after FolR1-TCB treatment with mean andstandard deviations. As comparison, PBMC from healthy donors wereco-cultured with the Skov3 tumor cell line and stimulated withFolR1-TCB. FIG. 12N depicts IFN-γ, IL-2, TNF and perforin in the cellculture supernatants as determined by Cytometric Bead Array or ELISA andnormalized to the amount of 1×10⁵ CD3⁺ T-cells (IFN-γ, TNF, IL-2) orCD3⁺ CD8⁺ T-cells (perforin) in the culture. FIG. 12O shows thatFolR1-TCB-induced tumor cell killing varies largely in tumor digests andmalignant effusions. FolR1 positive and negative tumor digests,malignant effusions or PBMCs from healthy donors were co-cultured withexogenously added fluorescently labeled FolR1⁺ Skov3 cells at an E:Tratio of 1:1 for 24 h in the presence or absence of FolR1-TCB. TheFolR1-TCB-induced specific killing of the Skov3 cells was determined byflow cytometry by measuring activated caspase 3 and the live/dead markerLIVE/DEAD®-near-IR. FolR1-TCB-mediated killing was calculated asfollows: % specific killing=100−[(% of Skov3 live cells in FolR1-TCBtreated sample/% of Skov3 live cells in untreated sample)×100]. FACSplots show FolR1-TCB-induced killing in a representative patient. Thep-values were calculated using the unpaired Mann-Whitney test.

FIGS. 13A-C show that FolR1-TCB-induced T cell activation shows nocorrelation with E:T ratio (FIG. 13A) or the amount of FolR1⁺ tumorcells (FIG. 13B). Tumor digests or malignant effusions were cultured for24 h in the presence or absence of FolR1-TCB. The FolR1-TCB inducedexpression of CD25 was correlated to E:T ratio or the amount of targetcells. MFI: mean fluorescence intensity.

FIGS. 14A-L show FolR1-TCB induced T cell activation inverselycorrelates with expression of PD-1 and Tim-3. Tumor digests or malignanteffusions were cultured for 24 h in the presence or absence ofFolR1-TCB. The expression of activation markers or markers of T cellfunction on CD8⁺ T cells was determined by flow cytometry. The FolR1-TCBinduced expression of CD25 (FIG. 4A-C), CD137 (FIG. 14D-F), ICOS (FIG.14G-I) and granzyme B (FIG. 14J-L) was correlated to baseline single- orco-expression of the inhibitory receptors PD-1 and Tim-3.

FIGS. 15A-C show FolR1-TCB induced IL-2 secretion inversely correlateswith co-expression of PD-1 and Tim-3. Tumor digests or malignanteffusions were cultured for 24 h in the presence or absence of FolR1TCB. IL-2 in the cell culture supernatants was determined by ELISA andnormalized to the amount of T cells. The FolR1 TCB induced IL-2secretion was correlated to baseline single- or co-expression of theinhibitory receptors PD-1 and Tim-3.

FIGS. 16A-F show FolR1-TCB induced tumor cell killing inverselycorrelates with co-expression of PD-1 and Tim-3. Tumor digests ormalignant effusions were co-cultured with exogenously added fluorescencelabelled Skov3 cells at a T cell to target cell ratio of 1:1 for 24 h inthe presence or absence of FolR1 TCB. The FolR1-TCB specific killing ofthe Skov3 cells was determined by flow cytometry by measuring activatedcaspase 3 and the live/dead marker Live/Dead-near-IR. The specifickilling was correlated to baseline single or co-expression of theinhibitory receptors PD-1, Tim-3 and CTLA-4.

FIGS. 17A-H show activation of tumor-infiltrating CD8⁺ T cells uponexposure to catumaxomab. Tumor digests or malignant effusions werecultured for 24 h in the presence or absence of catumaxomab. (FIG.17A-D) The expression of activation markers or markers of T cellfunction on CD8⁺ T cells was determined by flow cytometry. (FIG. 17E-H)Graphs showing the baseline expression of inhibitory receptors.

FIGS. 18A-R show Catumaxomab-induced T cell activation inverselycorrelates with co-expression of inhibitory receptors. Tumor digests ormalignant effusions were cultured for 24 h in the presence or absence ofcatumaxomab. T cell activation and effector functions were correlated tothe expression of PD-1 (FIG. 18A-F), Tim-3 (FIG. 18G-L) or of thecombination of PD-1 and Tim-3 (FIG. 18M-R).

FIGS. 19A-H show expression of inhibitory receptors ontumor-infiltrating T cells in Non-small cell lung cancer patients. CD8⁺and CD4⁺ T cells in tumor samples were characterized by flow cytometryfor their expression of inhibitory receptors (FIG. 19A-F). FIG. 19Gshows the gating strategy for one representative donor. FIG. 19H showsresults of analysis and heat mapping of indicated cell subsets based onthe percentage of expression, with the use of an Excel conditionalformatting program.

FIGS. 20A-E show T cell activation and effector functions uponpolyclonal stimulation by CD3/CD28 antibodies. Expression of CD25 andGranzyme B (FIG. 20A-B) as well as IL-2, IFN-γ and TNF-α (FIG. 20C-E) asmarkers for T cell activation and effector function, respectively, wasanalyzed in T cells from digested tumor samples after stimulation ofwhole tumor digests with agonistic CD3 and CD28 antibodies.

FIGS. 21A-N show expression of inhibitory receptors and T celldysfunction. Expression of CD25 and Granzyme B (FIG. 21A-B) as well asIL-2, IFN-γ and TNF-α (FIG. 21C-E) upon polyclonal stimulation by ananti-CD3/anti-CD28 antibodies correlates with the cumulative expressionof inhibitory receptors indicated by the iR Score. FIG. 21F shows anexemplary calculation of iR scores. The percentage of expression ofPD-1, Tim-3, CTLA-4, LAG-3 and BTLA was analyzed in all NSCLC samplesand the median as well as interquartile ranges were determined. For thecalculation of the iR score each patient received points for theexpression of each of the determined inhibitory receptors based on thequartile within which the expression coincided. A maximum of 15 pointscould be reached; the calculated score of each sample was normalized tothis maximum amount of points. FIG. 21G-K show expression of inhibitoryreceptors increases with tumor stage. Expression of inhibitory receptorson CD8⁺ tumor infiltrating T-cells was correlated to the TNM stage. FIG.21L-N show increased cumulative expression of inhibitory receptors withtumor progression. The cumulative expression of the inhibitory receptorsPD-1, Tim-3, CTLA-4, LAG-3 and BTLA, as represented by the iR score, wascorrelated to the nodal status and the TNM stage.

FIGS. 22A-I show expression of PD-1 and Tim-3 correlates with T celldysfunction. Expression of CD25 and Granzyme B (FIG. 22A-C) as well asIL-2, IFN-γ and TNF-α (FIG. 22D-F) upon polyclonal stimulation byCD3/CD28 correlates with the expression of PD-1 (FIG. 22A-C), Tim-3(FIG. 22D-F) or PD-Tim-3 (FIG. 22G-I) on tumor-infiltrating T cells.

FIGS. 23A-E show that the effect of PD-1 or combined PD-1/Tim-3 blockadevaries between patients. Digests were stimulated by agonisticanti-CD3/anti-CD28 antibodies with the addition of blocking antibodiesto PD-1 alone or in combination with Tim-3. Secretion of IFN-γ, TNF-αand IL-2 was determined by ELISA and normalized to 1×10⁶ T cells. FIG.23A-C show T cells from a patient where T cell function can be rescuedby addition of blocking Abs (BS-268) and T cells from a patient with noresponse to PD-1 or PD-1/Tim-3 blockade. The difference in expression([% expression Ab treated]-[% expression untreated]) is shown. FIG. 23Dshows respective flow cytometry plots with PD-1^(hi) and PD-1^(int)subsets. FIG. 23E shows a summary of IL-2, TNF-α and IFN-γ secretion byT cells from six patients, as determined by ELISA and normalized to1×10⁶ CD3⁺ T cells.

FIGS. 24A-F show that the effect of PD-1 or combined PD-Tim-3 blockadediffers in PD-1^(hi) and PD-1^(int) subsets. Correlation of the increasein cytokine production by PD-1 or combined PD-1/Tim-3 blockade withPD-1^(hi) and PD-1^(int) subsets are indicated by PD-1^(hi)/PD-1^(int)ratio.

FIGS. 25A-I show activation of CD4⁺ T cells in tumor digests andmalignant effusions upon exposure to FolR1-TCB. Tumor digests ormalignant effusions were cultured for 24 h in the presence or absence ofFolR1-TCB or the control TCB DP-47. The expression of activation markersor markers of T cell function on CD8⁺ T cells was determined by flowcytometry.

FIGS. 26A-C show FolR1-TCB induced T cell activation is independent ofCTLA-4, Lag-3 and BTLA expression. Tumor digests or malignant effusionswere cultured for 24 h in the presence or absence of FolR1-TCB. Theexpression of CD25 on CD8⁺ T cells was determined by flow cytometry. TheFolR1-TCB induced expression of CD25 was correlated to baselineexpression of CTLA-4, Lag-3 and BTLA.

FIGS. 27A-C show FolR1-TCB induces cytokine secretion only in patientswith a low percentage of PD-1^(h) expressing CD8⁺ T cells. Tumor digestsor malignant effusions were cultured for 24 h in the presence or absenceof FolR1-TCB. IFN-γ, TNF and IL-2 in the cell culture supernatants wasdetermined and normalized to the amount of 1×10⁵ T cells in the culture.The FolR1-TCB induced cytokine secretion was correlated to baselinePD-1^(hi) expression.

FIGS. 28A-F show that treatment with a PD-blocking antibody fails toinduce cytokine secretion in tumor digests or malignant effusions frompatients with lung and ovarian cancer with a low percentage of PD-1^(hi)expressing cells. Tumor digests or malignant effusions were cultured for24 h with FolR1-TCB in the presence or absence of PD-1 blocking antibody(FIG. 28A-C) or the combination of PD-1 and Tim-3 blocking antibodies(FIG. 28D-F). IFN-γ, TNF and IL-2 in the cell culture supernatants wasdetermined and normalized to the amount of 1×10⁵ T cells in the culture.The cytokine secretion induced by the blocking antibodies compared toFolR1-TCB treatment alone was correlated to baseline PD-1^(hi)expression.

FIGS. 29A-B show results from a FACS based internalization assay. Thedata show that the Fab fragment (<TIM-3> Fab) of anti-TIM3 antibodyTim3_0022 (abbreviated as <TIM-3> Ab(022)) internalized into rec CHOK1cells expressing huTIM-3 after incubation at 37° C. with similar kineticas the antibody in the full IgG format.

FIGS. 30A-B show binding of anti-TIM3 antibodies to RPMI-8226 cells(antibody desigantion clone 0016 refers to antibody Tim3_0016, clone0016 refers to antibody Tim3_0016 variant (antibody Tim3_0018), clone0022 refers to antibody Tim3_00122, etc.). FIG. 30B shows binding ofanti-TIM3 antibodies to Pfeiffer cells (antibody designation clone 0016refers to antibody Tim3_0016, clone 0016 refers to antibody Tim3_0016variant (antibody Tim3_0018), clone 0022 refers to antibody Tim3_00122,etc.).

FIG. 31 shows expression level of TIM-3 on different patient AML cellsamples by FACS using anti-TIM-3 mAbs.

FIG. 32 shows a heat map of expression of inhibitory receptors on NSCLCassociated TILs. Co-expression of inhibitory receptors ontumor-infiltrating CD8⁺ T-cells positive for the indicated immunecheckpoint is shown as a heat map displaying the percentage ofexpression for the additional receptors.

FIG. 33 shows a radar plot of expression of inhibitory receptors onNSCLC associated TILs. Co-expression of inhibitory receptors ontumor-infiltrating CD8⁺ T-cells positive for the indicated immunecheckpoint is shown as a radar plot indicating the mean expression andstandard deviation of the four other receptors.

FIGS. 34A-D show the percentage of PD-1^(hi) or PD-1^(int) CD8⁺ T cellsexpressing additional immune checkpoints. Each dot represents onepatient samples. The p values were calculated using the Wilcoxon ranksum test.

FIGS. 35A-F show intratumoral T cell inhibitory receptor expression andT cell function. FIG. 35A shows the gating strategy for identificationof PD-1^(hi), PD-1^(int), and PD-1^(neg) CD8⁺ subsets of T-cells fromtwo representative patients. FIG. 35B shows distribution of inidicated Tcell subsets in the tumor samples analyzed. FIG. 35C shows that T-cellfunctions induced by anti-CD3/-CD28 stimulation depend on the PD-1expression level of CD8⁺ T-cells. Tumor digests and malignant effusionswere cultured for 24 h in the presence or absence of agonisticanti-CD3/-CD28 antibodies. The increase in the expression of CD25 onCD8⁺ T-cells (FIG. 35C) and the increase in the effector cytokinesIFN-γ, IL-2, and TNF (FIG. 35D) were determined in PD-1^(hi) scarce andabundant tumors. p-values were calculated using the unpairedMann-Whitney test. Tumor samples were divided according to thepercentage of PD-1^(hi) expressing CD8⁺ cells in two groups withPD-1^(hi) scarce and abundant expression, respectively (FIG. 35E). Theexpression level of the inhibitory receptors PD-1, Tim-3, CTLA-4, Lag-3,and BTLA was determined by flow cytometry on CD8⁺ T-cells from tumordigests or malignant effusions (FIG. 35F).

FIGS. 36A-E show patterns of inhibitory receptor expression andpercentage of scarce and abundant CD8⁺ T-cells. FIG. 36A-D showco-expression of Tim-3, CTLA-4, Lag-3, and BTLA on PD-1^(hi),PD-1^(int), and PD-1^(neg) CD8⁺ T-cells. The p-values were calculatedusing one-way ANOVA with Bonferroni post-hoc-test. FIG. 36E: FolR1⁺tumor samples were divided according to the percentage of PD-1^(hi)expressing CD8⁺ cells in two groups with PD-1^(hi) scarce and abundantexpression, respectively.

FIGS. 37A-H show that FolR1-TCB-induced T-cell functions depend on thePD-1 expression level of CD8⁺ T-cells. FolR1⁺ tumor digests andmalignant effusions were cultured for 24 h in the presence or absence ofFolR1-TCB. The increase in the expression of activation markers on CD8⁺T-cells (FIG. 37A-C) and the increase in the effector cytokines IFN-γ,IL-2, TNF, and perforin (FIG. 37D-G) was determined in PD-1^(hi) scarceand abundant tumors. FIG. 37H shows target cell killing. Both FolR1positive and negative tumor samples were adjusted by addition of theFolR1⁺ Skov3 cell line to an E:T ratio of 1:1 and killing was comparedin PD-1^(hi) scarce and abundant tumors. p-values were calculated usingthe unpaired Mann-Whitney test.

FIGS. 38A-E show that PD-1 blockade increases cytokine production butnot their cytolytic function in T-cells from PD-1^(hi) scarce tumorsonly. FIG. 38A-D: FolR1⁺ tumor digests or malignant effusions werecultured for 24 h with FolR1-TCB in the presence or absence of a PD-1blocking antibody. IFN-γ, IL-2, TNF, and perforin in the cell culturesupernatants were determined by Cytometric Bead Array or ELISA andnormalized to the amount of 1×10⁵ CD3⁺ T-cells (IFN-γ, IL-2, TNF, FIG.38A-C) or CD3⁺CD8⁺ T-cells (perforin, FIG. 38D). The increase incytokine secretion upon combined FolR1-TCB and anti-PD-1 treatmentcompared with FolR1-TCB alone was determined in PD-1^(hi) scarce andabundant tumors. FIG. 38E: Tumor digests or malignant effusions wereco-cultured with exogenously added fluorescently labeled Skov3 cells atan E:T ratio of 1:1 for 24 h in the presence or absence of a PD-1blocking antibody and FolR1-TCB. The increase in specific killing by theanti-PD-1 antibody was compared in PD-1^(hi) scarce and abundant tumors.p-values were calculated using the unpaired Mann-Whitney test.

FIG. 39 shows detailed patient characteristics.

FIGS. 40A-C show activation of CD8⁺ T-cells upon exposure to increasingconcentrations of FolR1-TCB. PBMCs were co-cultured with Skov3 cells for24 h in the presence or absence of FolR1-TCB or the unspecific controlDP-47-TCB. FIG. 40A shows the expression of FolR1 on Skov3. Shadedhistogram: isotype control; open histogram: anti-FolR1-antibody. FIG.40B: The expression of the activation markers CD25, CD137, and ICOS onCD8⁺ T-cells was determined by flow cytometry. FIG. 40C: IFN-v. IL-2,and TNF in the cell culture supernatants were determined by ELISA andnormalized to the amount of 1×10⁵ CD3⁺ T-cells.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “A bispecific antibody that specifically binds Folate Receptor1 (FolR1) and CD3,” “T cell activating bispecific antigen bindingmolecule specific for FolR1 and CD3” and “FolR1 TCB” are usedinterchangeably herein and refer to a bispecific antibody that iscapable of binding FolR1 and CD3 with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingCD3⁺ T cells to FolR2⁺ target cells.

The terms “anti-TIM3 antibody” and “TIM3 antibody” are used synonymouslyherein to refer to an antibody that specifically binds to TIM3. Ananti-TIM3 antibody described herein refers to an antibody that iscapable of binding TIM3, especially a TIM3 polypeptide expressed on acell surface, with sufficient affinity such that the antibody is usefulas a diagnostic and/or therapeutic agent. In one embodiment, the extentof binding of an antibody that specifically binds TIM3 to an unrelatednon-TIM3 protein is less than about 10% of the binding of the antibodyto TIM3 as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry(FACS). In certain embodiments, an antibody that specifically binds TIM3has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g. from 10⁻⁸ M to10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, anantibody that specifically binds TIM3 binds to an epitope of TIM3 thatis conserved among DR5 from different species. Preferably said antibodybinds to human and cynomolgus monkey TIM3. The term “An antibody thatspecifically binds TIM3” also encompasses bispecific antibodies that arecapable of binding TIM3 and a second antigen.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies, cross-Fab fragments; linear antibodies; single-chainantibody molecules (e.g. scFv); and multispecific antibodies formed fromantibody fragments. scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibodyfragments comprise single chain polypeptides having the characteristicsof a VH domain, namely being able to assemble together with a VL domain,or of a VL domain, namely being able to assemble together with a VHdomain to a functional antigen binding site and thereby providing theantigen binding property of full length antibodies.

As used herein, “Fab fragment” refers to an antibody fragment comprisinga light chain fragment comprising a VL domain and a constant domain of alight chain (CL), and a VH domain and a first constant domain (CH1) of aheavy chain. In one embodiment the bispecific antibodies of theinvention comprise at least one Fab fragment, wherein either thevariable regions or the constant regions of the heavy and light chainare exchanged. Due to the exchange of either the variable regions or theconstant regions, said Fab fragment is also referred to as “cross-Fabfragment” or “xFab fragment” or “crossover Fab fragment”. Two differentchain compositions of a crossover Fab molecule are possible andcomprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable region (VL) and the heavy chainconstant region (CH1), and a peptide chain composed of the heavy chainvariable region (VH) and the light chain constant region (CL). Thiscrossover Fab molecule is also referred to as CrossFab _((VLVH)). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the light chainconstant region (CL), and a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1). Thiscrossover Fab molecule is also referred to as CrossFab _((CLCH1)).Bispecific antibody formats comprising crossover Fab fragments have beendescribed, for example, in WO 2009/080252, WO 2009/080253, WO2009/080251, WO 2009/080254, WO 2010/136172, WO 2010/145792 and WO2013/026831.

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction:

a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide ofat least 30 amino acids, preferably between 32 and 50 amino acids. Saidsingle chain Fab fragments a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 and d) VL-CH1-linker-VH-CL,are stabilized via the natural disulfide bond between the CL domain andthe CH1 domain. In addition, these single chain Fab molecules might befurther stabilized by generation of interchain disulfide bonds viainsertion of cysteine residues (e.g. position 44 in the variable heavychain and position 100 in the variable light chain according to Kabatnumbering). The term “N-terminus denotes the last amino acid of theN-terminus. The term “C-terminus denotes the last amino acid of theC-terminus. By “fused” or “connected” is meant that the components (e.g.a Fab molecule and an Fc domain subunit) are linked by peptide bonds,either directly or via one or more peptide linkers.

The term “linker” as used herein refers to a peptide linker and ispreferably a peptide with an amino acid sequence with a length of atleast 5 amino acids, preferably with a length of 5 to 100, morepreferably of 10 to 50 amino acids. In one embodiment said peptidelinker is (G_(x)S)_(n) (SEQ ID NOS 384 and 385) or (G_(x)S)_(n)G_(m)(SEQ ID NOS 429 and 430) with G=glycine, S=serine, and (x=3, n=3, 4, 5or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3),preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In oneembodiment said peptide linker is (G₄S)₂ (SEQ ID NO: 386).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and β₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “antigen binding domain” refers to the part of an antigenbinding molecule that comprises the area which specifically binds to andis complementary to part or all of an antigen. Where an antigen islarge, an antigen binding molecule may only bind to a particular part ofthe antigen, which part is termed an epitope. An antigen binding domainmay be provided by, for example, one or more antibody variable domains(also called antibody variable regions). Preferably, an antigen bindingdomain comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species, usually prepared by recombinant DNAtechniques. Chimeric antibodies comprising a rabbit variable region anda human constant region are preferred. Other preferred forms of“chimeric antibodies” encompassed by the present invention are those inwhich the constant region has been modified or changed from that of theoriginal antibody to generate the properties according to the invention,especially in regard to C1q binding and/or Fc receptor (FcR) binding.Such chimeric antibodies are also referred to as “class-switchedantibodies”. Chimeric antibodies are the product of expressedimmunoglobulin genes comprising DNA segments encoding immunoglobulinvariable regions and DNA segments encoding immunoglobulin constantregions. Methods for producing chimeric antibodies involve conventionalrecombinant DNA and gene transfection techniques are well known in theart. See e.g. Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependentcellular phagocytosis (ADCP), cytokine secretion, immunecomplex-mediated antigen uptake by antigen presenting cells; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991. A “subunit”of an Fc domain as used herein refers to one of the two polypeptidesforming the dimeric Fc domain, i.e. a polypeptide comprising C-terminalconstant regions of an immunoglobulin heavy chain, capable of stableself-association. For example, a subunit of an IgG Fc domain comprisesan IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. As also mentioned forchimeric and humanized antibodies according to the invention the term“human antibody” as used herein also comprises such antibodies which aremodified in the constant region to generate the properties according tothe invention, especially in regard to C1q binding and/or FcR binding,e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. fromIgG1 to IgG4 and/or IgG1/IgG4 mutation.)

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

The term “hypervariable region” or “HVR,” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (12), 91-96 (13), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably in reference toportions of the variable region that form the antigen binding regions.This particular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table A as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding a bispecific antibody that specificallybinds DR5 and FAP antibody” refers to one or more nucleic acid moleculesencoding antibody heavy and light chains (or fragments thereof),including such nucleic acid molecule(s) in a single vector or separatevectors, and such nucleic acid molecule(s) present at one or morelocations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Forexample, the anti-PD-L1 antibodies of the invention block the signalingthrough PD-1 so as to restore a functional response by T-cells (e.g.,proliferation, cytokine production, target cell killing) from adysfunctional state to antigen stimulation.

An “agonist” or activating antibody is one that enhances or initiatessignaling by the antigen to which it binds. In some embodiments, agonistantibodies cause or activate signaling without the presence of thenatural ligand.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“No substantial cross-reactivity” means that a molecule (e.g., anantibody) does not recognize or specifically bind an antigen differentfrom the actual target antigen of the molecule (e.g. an antigen closelyrelated to the target antigen), particularly when compared to thattarget antigen. For example, an antibody may bind less than about 10% toless than about 5% to an antigen different from the actual targetantigen, or may bind said antigen different from the actual targetantigen at an amount consisting of less than about 10%, 9%, 8% 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%,1%, or 0.5%, and most preferably less than about 0.2% or 0.1% antigendifferent from the actual target antigen.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “PD-1 axis binding antagonist” is a molecule that inhibits theinteraction of a PD-1 axis binding partner with either one or more ofits binding partner, so as to remove T-cell dysfunction resulting fromsignaling on the PD-1 signaling axis—with a result being to restore orenhance T-cell function {e.g., proliferation, cytokine production,target cell killing). As used herein, a PD-1 axis binding antagonistincludes a PD-1 binding antagonist, a PD-L1 binding antagonist and aPD-L2 binding antagonist.

The term “PD-1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to itsbinding partners. In a specific aspect, the PD-1 binding antagonistinhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1binding antagonists include anti-PD-1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-1 withPD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reducesthe negative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-1 soas render a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, thePD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect,a PD-1 binding antagonist is MDX-1106 described herein. In anotherspecific aspect, a PD-binding antagonist is Merck 3745 described herein.In another specific aspect, a PD-1 binding antagonist is CT-01 1described herein.

The term “PD-L1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L 1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L 1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1 105 described herein.In still another specific aspect, an anti-PD-L1 antibody is MPDL3280Adescribed herein.

The term “PD-L2 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to itsbinding partners. In a specific aspect, the PD-L2 binding antagonistinhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2antagonists include anti-PD-L2 antibodies, antigen binding fragmentsthereof, immunoadhesins, fusion proteins, oligopeptides and othermolecules that decrease, block, inhibit, abrogate or interfere withsignal transduction resulting from the interaction of PD-L2 with eitherone or more of its binding partners, such as PD-1. In one embodiment, aPD-L2 binding antagonist reduces the negative co-stimulatory signalmediated by or through cell surface proteins expressed on T lymphocytesmediated signaling through PD-L2 so as render a dysfunctional T-cellless dysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, a PD-L2 binding antagonist is animmunoadhesin.

A “PD-1 oligopeptide” “PD-L1 oligopeptide” or “PD-L2 oligopeptide” is anoligopeptide that binds, preferably specifically, to a PD-1, PD-L1 orPD-L2 negative costimulatory polypeptide, respectively, including areceptor, ligand or signaling component, respectively, as describedherein. Such oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. Such oligopeptides are usually at least about 5amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100amino acids in length or more. Such oligopeptides may be identifiedusing well known techniques. In this regard, it is noted that techniquesfor screening oligopeptide libraries for oligopeptides that are capableof specifically binding to a polypeptide target are well known in theart (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT PublicationNos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci.U.S.A., 81:3998-4002 (1984); Geysen et al, Proc. Natl. Acad. Sci.U.S.A., 82: 178-182 (1985); Geysen et al, in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Metk, 102:259-274(1987); Schoofs et al., J. Immunol., 140:61 1-616 (1988), Cwirla, S. E.et al. Proc. Natl. Acad. Sci. USA, 87:6378 (1990); Lowman, H. B. et al.Biochemistry, 30: 10832 (1991); Clackson, T. et al. Nature, 352: 624(1991); Marks, J. D. et al., J. Mol. Biol., 222:581 (1991); Kang, A. S.et al. Proc. Natl. Acad. Sci. USA, 88:8363 (1991), and Smith, G. P.,Current Opin. Biotechnol, 2:668 (1991).

The term “anergy” refers to the state of unresponsiveness to antigenstimulation resulting from incomplete or insufficient signals deliveredthrough the T-cell receptor (e.g. increase in intracellular Ca⁺² in theabsence of ras-activation). T cell anergy can also result uponstimulation with antigen in the absence of co-stimulation, resulting inthe cell becoming refractory to subsequent activation by the antigeneven in the context of costimulation. The unresponsive state can oftenbe overriden by the presence of lnterleukin-2. Anergic T-cells do notundergo clonal expansion and/or acquire effector functions.

The term “exhaustion” refers to T cell exhaustion as a state of T celldysfunction that arises from sustained TCR signaling that occurs duringmany chronic infections and cancer. It is distinguished from anergy inthat it arises not through incomplete or deficient signaling, but fromsustained signaling. It is defined by poor effector function, sustainedexpression of inhibitory receptors and a transcriptional state distinctfrom that of functional effector or memory T cells. Exhaustion preventsoptimal control of infection and tumors. Exhaustion can result from bothextrinsic negative regulatory pathways (e.g., immunoregulatorycytokines) as well as cell intrinsic negative regulatory (costimulatory)pathways (PD-1, B7-H3, B7-H4, etc.).

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells,increased proliferation, increased antigen responsiveness (e.g., viral,pathogen, or tumor clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.[0046] “Immunogenecity” refers to the ability of a particular substanceto provoke an immune response. Tumors are immunogenic and enhancingtumor immunogenicity aids in the clearance of the tumor cells by theimmune response. Examples of enhancing tumor immunogenicity includetreatment with anti-PDL antibodies and a ME inhibitor.

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

The term “Fibroblast activation protein (FAP)”, as used herein, refersto any native FAP from any vertebrate source, including mammals such asprimates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessed FAPas well as any form of FAP that results from processing in the cell. Theterm also encompasses naturally occurring variants of FAP, e.g., splicevariants or allelic variants. Preferably, an anti-FAP antibody of theinvention binds to the extracellular domain of FAP. The amino acidsequence of exemplary FAP polypeptide sequences, including the sequenceof human FAP, are disclosed in WO 2012/020006.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term cancer as used herein refers to proliferative diseases, such asthe cancer is colorectal cancer, sarcoma, head and neck cancer, squamouscell carcinoma, breast cancer, pancreatic cancer, gastric cancer,non-small-cell lung carcinoma, small-cell lung cancer and mesothelioma,including refractory versions of any of the above cancers, or acombination of one or more of the above cancers. In one embodiment, thecancer is colorectal cancer and optionally the chemotherapeutic agent isIrinotecan. In embodiments in which the cancer is sarcoma, optionallythe sarcoma is chondrosarcoma, leiomyosarcoma, gastrointestinal stromaltumours, fibrosarcoma, osteosarcoma, liposarcoma or malignant fibroushistiocytoma.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As usedherein, the term “antigen binding molecule” refers in its broadest senseto a molecule that specifically binds an antigenic determinant. Examplesof antigen binding molecules are immunoglobulins and derivatives, e.g.fragments, thereof.

The term “antigen-binding site of an antibody” when used herein refer tothe amino acid residues of an antibody which are responsible forantigen-binding. The antigen-binding portion of an antibody comprisesamino acid residues from the “complementary determining regions” or“CDRs”. “Framework” or “FR” regions are those variable domain regionsother than the hypervariable region residues as herein defined.Therefore, the light and heavy chain variable domains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding and defines the antibody'sproperties. CDR and FR regions are determined according to the standarddefinition of Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991) and/or those residues from a “hypervariable loop”.

Antibody specificity refers to selective recognition of the antibody fora particular epitope of an antigen. Natural antibodies, for example, aremonospecific. “Bispecific antibodies” according to the invention areantibodies which have two different antigen-binding specificities.Antibodies of the present invention are specific for two differentantigens, i.e. DR5 as first antigen and FAP as second antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises at least twoantigen binding sites, each of which is specific for a differentantigenic determinant. In certain embodiments the bispecific antigenbinding molecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The antibody provided herein is a multispecific antibody, e.g. abispecific antibody. Multispecific antibodies are monoclonal antibodiesthat have binding specificities for at least two different sites.Provided herein is a bispecific antibody, with binding specificities forFAP and DR5. In certain embodiments, bispecific antibodies may bind totwo different epitopes of DR5. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express DR5. Bispecificantibodies can be prepared as full length antibodies or antibodyfragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising at least one antigen binding site that binds to FAP orDR5 as well as another, different antigen (see, US 2008/0069820, forexample).

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antibody molecule.As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denotethe presence of two binding sites, four binding sites, and six bindingsites, respectively, in an antibody molecule. The bispecific antibodiesaccording to the invention are at least “bivalent” and may be“trivalent” or “multivalent” (e.g. “tetravalent” or “hexavalent”).

Antibodies of the present invention have two or more binding sites andare bispecific. That is, the antibodies may be bispecific even in caseswhere there are more than two binding sites (i.e. that the antibody istrivalent or multivalent). Bispecific antibodies of the inventioninclude, for example, multivalent single chain antibodies, diabodies andtriabodies, as well as antibodies having the constant domain structureof full length antibodies to which further antigen-binding sites (e.g.,single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2) arelinked via one or more peptide-linkers. The antibodies can be fulllength from a single species, or be chimerized or humanized.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transfectants” and “transfected cells” include theprimary subject cell and cultures derived there from without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen in an in-vitroassay, preferably in a surface plasmon resonance assay (SPR, BIAcore,GE-Healthcare Uppsala, Sweden). The affinity of the binding is definedby the terms ka (rate constant for the association of the antibody fromthe antibody/antigen complex), kD (dissociation constant), and KD(kD/ka). Binding or specifically binding means a binding affinity (KD)of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to 10⁻¹³ mol/l.

Binding of the antibody to the death receptor can be investigated by aBIAcore assay (GE-Healthcare Uppsala, Sweden). The affinity of thebinding is defined by the terms ka (rate constant for the association ofthe antibody from the antibody/antigen complex), kD (dissociationconstant), and KD (kD/ka)

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. The T cell activating bispecific antigen bindingmolecules of the invention are capable of inducing T cell activation.Suitable assays to measure T cell activation are known in the artdescribed herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Inparticular “target cell antigen” refers to Folate Receptor 1.

As used herein, the terms “first” and “second” with respect to antigenbinding moieties etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of the T cellactivating bispecific antigen binding molecule unless explicitly sostated.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins referred to as antigens herein,e.g., FolR1 and CD3, can be any native form the proteins from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g. mice and rats), unless otherwise indicated. In aparticular embodiment the antigen is a human protein. Where reference ismade to a specific protein herein, the term encompasses the“full-length”, unprocessed protein as well as any form of the proteinthat results from processing in the cell. The term also encompassesnaturally occurring variants of the protein, e.g. splice variants orallelic variants. Exemplary human proteins useful as antigens include,but are not limited to: FolR1 (Folate receptor alpha (FRA); Folatebinding protein (FBP); human FolR1 UniProt no.: P15328; murine FolR1UniProt no.: P35846; cynomolgus FolR1 UniProt no.: G7PR14) and CD3,particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence;or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, for thecynomolgus [Macaca fascicularis] sequence). The T cell activatingbispecific antigen binding molecule of the invention binds to an epitopeof CD3 or a target cell antigen that is conserved among the CD3 ortarget antigen from different species. In certain embodiments the T cellactivating bispecific antigen binding molecule of the invention binds toCD3 and FolR1, but does not bind to FolR2 (Folate receptor beta; FRB;human FolR2 UniProt no.: P14207) or FolR3 (Folate receptor gamma; humanFolR3 UniProt no.: P41439).

As used herein, the terms “engineer, engineered, engineering,”particularly with the prefix “glyco-,” as well as the term“glycosylation engineering” are considered to include any manipulationof the glycosylation pattern of a naturally occurring or recombinantpolypeptide or fragment thereof. Glycosylation engineering includesmetabolic engineering of the glycosylation machinery of a cell,including genetic manipulations of the oligosaccharide synthesispathways to achieve altered glycosylation of glycoproteins expressed incells. Furthermore, glycosylation engineering includes the effects ofmutations and cell environment on glycosylation. In one embodiment, theglycosylation engineering is an alteration in glycosyltransferaseactivity. In a particular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.

II. Compositions and Methods

In one aspect, the invention is based on the use of a therapeuticcombination of a T cell activating bispecific antigen binding molecule,e.g., a T cell activating bispecific antigen binding molecule comprisinga first antigen binding site specific for Folate Receptor 1 (FolR1) anda second antigen binding site specific for CD3, and a PD-1 axis bindingantagonist, e.g., for the treatment of cancer. In some embodiments thetherapeutic combination further includes a TIM3 antagonist.

A. Combination Therapies of a T Cell Activating Bispecific AntigenBinding Molecule and a PD-1 Axis Binding Antagonist

Broadly, the present invention relates to T cell activating bispecificantigen binding molecules and their use in combination with a PD-1 axisbinding antagonists. The advantage of the combination over monotherapyis that the T cell activating bispecific antigen binding molecules usedin the present invention enable re-direction and activation of T cellsto the targeted cell while the PD-1 axis binding antagonist enhances Tcell function by reducing T cell exhaustion.

In one aspect, provided herein is a method for treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of a T cell activating bispecific antigenbinding molecules, e.g., a FolR1-TCB, and a PD-1 axis bindingantagonist. In some embodiments, the treatment results in sustainedresponse in the individual after cessation of the treatment. The methodsof this invention may find use in treating conditions where enhancedimmunogenicity is desired such as increasing tumor immunogenicity forthe treatment of cancer. A variety of cancers may be treated, or theirprogression may be delayed, including but are not limited to a cancerthat may contain a BRAF V600E mutation, a cancer that may contain a BRAFwildtype, a cancer that may contain a KRAS wildtype, or a cancer thatmay contain an activating KRAS mutation.

In some embodiments, the individual has endometrial cancer. Theendometrial cancer may be at early stage or late state. In someembodiments, the individual has melanoma. The melanoma may be at earlystage or at late stage. In some embodiments, the individual hascolorectal cancer. The colorectal cancer may be at early stage or atlate stage. In some embodiments, the individual has lung cancer, e.g.,non-small cell lung cancer. The non-small cell lung cancer may be atearly stage or at late stage. In some embodiments, the individual haspancreatic cancer. The pancreatic cancer may be at early stage or latestate. In some embodiments, the individual has a hematologicalmalignancy. The hematological malignancy may be early stage or latestage. In some embodiments, the individual has ovarian cancer. Theovarian cancer may be at early stage or at late stage. In someembodiments, the individual has breast cancer. The breast cancer may beat early stage or at late stage. In some embodiments, the individual hasrenal cell carcinoma. The renal cell carcinoma may be at early stage orat late stage.

In some embodiments, the individual is a mammal, such as domesticatedanimals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g.,humans and non-human primates such as monkeys), rabbits, and rodents(e.g., mice and rats). In some embodiments, the individual treated is ahuman.

In another aspect, provided herein is a method of enhancing immunefunction in an individual having cancer comprising administering aneffective amount of a T cell activating bispecific antigen bindingmolecules, specifically, a FolR1-TCB, and a PD-1 axis bindingantagonist.

In some embodiments, the T cells in the individual have enhancedpriming, activation, proliferation and/or effector function relative toprior to the administration of the T cell activating bispecific antigenbinding molecules and the PD-1 pathway antagonist. In some embodiments,the T cell effector function is secretion of at least one of IL-2, IFN-γand TNF-α. In one embodiment, administering a FolR1-TCB and ananti-PDL-1 antibody results in increased T cell secretion of IL-2, IFN-γand TNF-α. In some embodiments, the T cell is a CD8+ T cell. In someembodiments, the T cell priming is characterized by elevated CD44expression and/or enhanced cytolytic activity in CD8 T cells. In someembodiments, the CD8 T cell activation is characterized by an elevatedfrequency of γ-IFT{circumflex over ( )}T CD8 T cells. In someembodiments, the CD8 T cell is an antigen-specific T-cell. In someembodiments, the immune evasion by signaling through PD-L1 surfaceexpression is inhibited. In some embodiments, the cancer has elevatedlevels of T-cell infiltration.

In some embodiments, the combination therapy of the invention comprisesadministration of a FolR1-TCB and a PD-1 axis binding antagonist. TheFolR1-TCB and a PD-1 axis binding antagonist may be administered in anysuitable manner known in the art. For example, FolR1-TCB and a PD-1 axisbinding antagonist may be administered sequentially (at different times)or concurrently (at the same time). In some embodiments, the FolR1-TCBis administered continuously. In some embodiments, the FolR1-TCB isadministered intermittently. In some embodiments, the FolR1-TCB isadministered before administration of the PD-1 axis binding antagonist.In some embodiments, the FolR1-TCB is administered simultaneously withadministration of the PD-1 axis binding antagonist. In some embodiments,the FolR1-TCB is administered after administration of the PD-1 axisbinding antagonist.

In some embodiments, provided is a method for treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of a T cell activating bispecific antigenbinding molecules, e.g., a FolR1-TCB, and a PD-1 axis bindingantagonist, further comprising administering an additional therapy.Specifically contemplated is an embodiment in which the additionaltherapy comprises a TIM-3 antagonist. Accordingly, in one aspect,provided herein is a method for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a T cell activating bispecific antigen bindingmolecules, specifically, a FolR1-TCB, a PD-1 axis binding antagonist,and a TIM-3 antagonist. Any TIM3 antagonist, e.g., those describedherein, can be used. The additional therapy may also be radiationtherapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, genetherapy, DNA therapy, viral therapy, R A therapy, immunotherapy, bonemarrow transplantation, nanotherapy, monoclonal antibody therapy, or acombination of the foregoing. The additional therapy may be in the formof adjuvant or neoadjuvant therapy. In some embodiments, the additionaltherapy is the administration of small molecule enzymatic inhibitor oranti-metastatic agent. In some embodiments, the additional therapy isthe administration of side-effect limiting agents (e.g., agents intendedto lessen the occurrence and/or severity of side effects of treatment,such as anti-nausea agents, etc.). In some embodiments, the additionaltherapy is radiation therapy. In some embodiments, the additionaltherapy is surgery. In some embodiments, the additional therapy is acombination of radiation therapy and surgery. In some embodiments, theadditional therapy is gamma irradiation. In some embodiments, theadditional therapy is therapy targeting P13K/A T/mTOR pathway, HSP90inhibitor, tubulin inhibitor, apoptosis inhibitor, and/orchemopreventative agent. The additional therapy may be one or more ofthe chemotherapeutic agents described hereabove.

T cell activating bispecific antigen binding molecules, e.g., aFolR1-TCB, and the PD-1 axis binding antagonist may be administered bythe same route of administration or by different routes ofadministration. In some embodiments, T cell activating bispecificantigen binding molecules, e.g., a FolR1-TCB is administeredintravenously, intramuscularly, subcutaneously, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally. In someembodiments, the PD-1 axis binding antagonist is administeredintravenously, intramuscularly, subcutaneously, topically, orally,transdermally, intraperitoneally, intraorbitally, by implantation, byinhalation, intrathecally, intraventricularly, or intranasally. Aneffective amount of the T cell activating bispecific antigen bindingmolecules and the PD-1 axis binding antagonist may be administered forprevention or treatment of disease. The appropriate dosage of the T cellactivating bispecific antigen binding molecules and/or the PD-1 axisbinding antagonist may be determined based on the type of disease to betreated, the type of the T cell activating bispecific antigen bindingmolecules and the PD-1 axis binding antagonist, the severity and courseof the disease, the clinical condition of the individual, theindividual's clinical history and response to the treatment, and thediscretion of the attending physician.

Any of the T cell activating bispecific antigen binding molecules, PD-1axis binding antagonists and the TIM-3 antagonists known in the art ordescribed below may be used in the methods.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a T cell activating bispecific antigen bindingmolecules as described herein, a PD-1 axis binding antagonists asdescribed herein and a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition further comprises a TIM3antagonist.

In a further aspect, the invention provides for a kit comprising a Tcell activating bispecific antigen binding molecule specific for FolateReceptor 1 (FolR1) and CD3, and a package insert comprising instructionsfor using the T cell activating bispecific antigen binding molecule witha PD-1 axis binding antagonist to treat or delay progression of cancerin an individual. In some embodiments, the kit further comprisesinstructions for using the T cell activating bispecific antigen bindingmolecule with a TIM3 antagonist. In a further aspect, the inventionprovides for a kit comprising a T cell activating bispecific antigenbinding molecule specific for Folate Receptor 1 (FolR1) and CD3 and aPD-1 axis binding antagonist, and a package insert comprisinginstructions for using the T cell activating bispecific antigen bindingmolecule and the PD-1 axis binding antagonist to treat or delayprogression of cancer in an individual. In one embodiment, the kitfurther comprises a TIM3 antagonist. In one of the embodiments, the PD-1axis binding antagonist is an anti-PD-1 antibody or ananti-PDL-antibody. In one embodiment, the PD-1 axis binding antagonistis an anti-PD-1 immunoadhesin.

In a further aspect, the invention provides a kit comprising:

(i) a first container comprising a composition which comprises a T cellactivating bispecific antigen binding molecule specific for FolateReceptor 1 (FolR1) and CD3 as described herein; and

(ii) a second container comprising a composition comprising a PD-1 axisbinding antagonist.

In a further aspect, the invention provides a kit comprising:

(i) a first container comprising a composition which comprises a T cellactivating bispecific antigen binding molecule specific for FolateReceptor 1 (FolR1) and CD3 as described herein;

(ii) a second container comprising a composition comprising a PD-1 axisbinding antagonist; and

(iii) a third container comprising a composition comprising a TIM3antagonist.

B. Exemplary T Cell Activating Bispecific Antigen Binding Molecule forUse in the Invention

The T cell activating bispecific antigen binding molecule of theinvention is bispecific, i.e. it comprises at least two antigen bindingmoieties capable of specific binding to two distinct antigenicdeterminants, i.e. to CD3 and to FolR1. According to the invention, theantigen binding moieties are Fab molecules (i.e. antigen binding domainscomposed of a heavy and a light chain, each comprising a variable and aconstant region). In one embodiment said Fab molecules are human. Inanother embodiment said Fab molecules are humanized. In yet anotherembodiment said Fab molecules comprise human heavy and light chainconstant regions.

The T cell activating bispecific antigen binding molecule of theinvention is capable of simultaneous binding to the target cell antigenFolR1 and CD3. In one embodiment, the T cell activating bispecificantigen binding molecule is capable of crosslinking a T cell and a FolR1expressing target cell by simultaneous binding to the target cellantigen FolR1 and CD3. In an even more particular embodiment, suchsimultaneous binding results in lysis of the FolR1 expressing targetcell, particularly a FolR1 expressing tumor cell. In one embodiment,such simultaneous binding results in activation of the T cell. In otherembodiments, such simultaneous binding results in a cellular response ofa T lymphocyte, particularly a cytotoxic T lymphocyte, selected from thegroup of: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers. In one embodiment, binding of the T cell activatingbispecific antigen binding molecule to CD3 without simultaneous bindingto the target cell antigen FolR1 does not result in T cell activation.

In one embodiment, the T cell activating bispecific antigen bindingmolecule is capable of re-directing cytotoxic activity of a T cell to aFolR1 expressing target cell. In a particular embodiment, saidre-direction is independent of MHC-mediated peptide antigen presentationby the target cell and and/or specificity of the T cell.

Particularly, a T cell according to some of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to CD3 (also referred to herein as an “CD3 antigen bindingmoiety” or “first antigen binding moiety”). In a particular embodiment,the T cell activating bispecific antigen binding molecule comprises notmore than one antigen binding moiety capable of specific binding to CD3.In one embodiment the T cell activating bispecific antigen bindingmolecule provides monovalent binding to CD3. In a particular embodimentCD3 is human CD3 or cynomolgus CD3, most particularly human CD3. In aparticular embodiment the CD3 antigen binding moiety is cross-reactivefor (i.e. specifically binds to) human and cynomolgus CD3. In someembodiments, the first antigen binding moiety is capable of specificbinding to the epsilon subunit of CD3 (see UniProt no. P07766 (version130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:150 for the human sequence;UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1, for thecynomolgus [Macaca fascicularis] sequence).

In some embodiments, the CD3 antigen binding moiety comprises at leastone heavy chain complementarity determining region (CDR) selected fromthe group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39and at least one light chain CDR selected from the group of SEQ ID NO:32, SEQ ID NO: 33, SEQ ID NO: 34.

In one embodiment the CD3 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, theheavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34.

In one embodiment the CD3 antigen binding moiety comprises a variableheavy chain comprising an amino acid sequence of: SEQ ID NO: 36 and avariable light chain comprising an amino acid sequence of: SEQ ID NO:31.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 36 and a light chain variable regionsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 31.

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to the target cell antigen FolR1 (also referred to herein as an“FolR1 binding moiety” or “second” or “third” antigen binding moiety).In one embodiment, the antigen binding moiety capable of binding to thetarget cell antigen FolR1 does not bind to FolR2 or FolR3. In aparticular embodiment the FolR1 antigen binding moiety is cross-reactivefor (i.e. specifically binds to) human and cynomolgus FolR1. In certainembodiments, the T cell activating bispecific antigen binding moleculecomprises two antigen binding moieties capable of binding to the targetcell antigen FolR1. In a particular such embodiment, each of theseantigen binding moieties specifically binds to the same antigenicdeterminant. In an even more particular embodiment, all of these antigenbinding moieties are identical. In one embodiment the T cell activatingbispecific antigen binding molecule comprises not more than two antigenbinding moieties capable of binding to FolR1.

The FolR1 binding moiety is generally a Fab molecule that specificallybinds to FolR1 and is able to direct the T cell activating bispecificantigen binding molecule to which it is connected to a target site, forexample to a specific type of tumor cell that expresses FolR1. In oneaspect the present invention provides a T cell activating bispecificantigen binding molecule comprising

-   -   (i) a first antigen binding moiety which is a Fab molecule        capable of specific binding to CD3, and which comprises at least        one heavy chain complementarity determining region (CDR)        selected from the group consisting SEQ ID NO: 37, SEQ ID NO: 38        and SEQ ID NO: 39 and at least one light chain CDR selected from        the group of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34; and    -   (ii) a second antigen binding moiety which is a Fab molecule        capable of specific binding to Folate Receptor 1 (FolR1).

In one embodiment the first antigen binding moiety which is a Fabmolecule capable of specific binding to CD3 comprises a variable heavychain comprising an amino acid sequence of SEQ ID NO: 36 and a variablelight chain comprising an amino acid sequence of SEQ ID NO: 31.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

-   -   (iii) a third antigen binding moiety which is a Fab molecule        capable of specific binding to FolR1.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

In one embodiment the T cell activating bispecific antigen bindingmolecule of any of the above embodiments additionally comprises an Fcdomain composed of a first and a second subunit capable of stableassociation.

In one embodiment the first antigen binding moiety and the secondantigen binding moiety are each fused at the C-terminus of the Fab heavychain to the N-terminus of the first or second subunit of the Fc domain.

In one embodiment the third antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety, optionally via a peptidelinker.

In a further particular embodiment, not more than one antigen bindingmoiety capable of specific binding to CD3 is present in the T cellactivating bispecific antigen binding molecule (i.e. the T cellactivating bispecific antigen binding molecule provides monovalentbinding to CD3).

T Cell Activating Bispecific Antigen Binding Molecule with a CommonLight Chain

The inventors of the present invention generated a bispecific antibodywherein the binding moieties share a common light chain that retains thespecificity and efficacy of the parent monospecific antibody for CD3 andcan bind a second antigen (e.g., FolR1) using the same light chain. Thegeneration of a bispecific molecule with a common light chain thatretains the binding properties of the parent antibody is notstraight-forward as the common CDRs of the hybrid light chain have toeffectuate the binding specificity for both targets. In one aspect thepresent invention provides a T cell activating bispecific antigenbinding molecule comprising a first and a second antigen binding moiety,one of which is a Fab molecule capable of specific binding to CD3 andthe other one of which is a Fab molecule capable of specific binding toFolR1, wherein the first and the second Fab molecule have identical VLCLlight chains. In one embodiment said identical light chain (VLCL)comprises the light chain CDRs of SEQ ID NO: 32, SEQ ID NO: 33 and SEQID NO: 34. In one embodiment said identical light chain (VLCL) comprisesSEQ ID NO. 35.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, theheavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 15 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 31 or variants thereofthat retain functionality.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO:15, and a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

(iii) a third antigen binding moiety (which is a Fab molecule) capableof specific binding to FolR1.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

Hence in one embodiment the present invention provides a T cellactivating bispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34.(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:18 and at least one light chain CDR selected from the group of SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, theheavy chain CDR3 of SEQ ID NO:18, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 15 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 15 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO:16, SEQ ID NO:402 and SEQ IDNO:400 and at least one light chain CDR selected from the group of SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34, and the FolR1 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO:16, the heavy chain CDR2 of SEQ ID NO:402,the heavy chain CDR3 of SEQ ID NO:400, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO:401 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:401 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 31 or variants thereofthat retain functionality.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO:401, and a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

(iii) a third antigen binding moiety (which is a Fab molecule) capableof specific binding to FolR1.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

Hence in one embodiment the present invention provides a T cellactivating bispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO:16, SEQ ID NO:402 and SEQ IDNO:400 and at least one light chain CDR selected from the group of SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) and which comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO:16, SEQ ID NO:402 and SEQ IDNO:400 and at least one light chain CDR selected from the group of SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO:16, the heavy chain CDR2 of SEQ ID NO:402, theheavy chain CDR3 of SEQ ID NO:400, the light chain CDR1 of SEQ ID NO:32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 ofSEQ ID NO:34.

In one embodiment the present invention provides a T cell activatingbispecific antigen binding molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a variable heavy chain comprising anamino acid sequence of SEQ ID NO: 36 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO:401 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO:401 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 31.

Thus, in one embodiment, the invention relates to bispecific moleculeswherein at least two binding moieties have identical light chains andcorresponding remodeled heavy chains that confer the specific binding tothe T cell activating antigen CD3 and the target cell antigen FolR1,respectively. The use of this so-called ‘common light chain’ principle,i.e. combining two binders that share one light chain but still haveseparate specificities, prevents light chain mispairing. Thus, there areless side products during production, facilitating the homogenouspreparation of T cell activating bispecific antigen binding molecules.

The components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1A-I and are furtherdescribed below.

In some embodiments, said T cell activating bispecific antigen bindingmolecule further comprises an Fc domain composed of a first and a secondsubunit capable of stable association. Below exemplary embodiments of Tcell activating bispecific antigen binding molecule comprising an Fcdomain are described.

T Cell Activating Bispecific Antigen Binding Molecule with a CrossoverFab Fragment

The inventors of the present invention generated a second bispecificantibody format wherein one of the binding moieties is a crossover Fabfragment. In one aspect of the invention a monovalent bispecificantibody is provided, wherein one of the Fab fragments of an IgGmolecule is replaced by a crossover Fab fragment. Crossover Fabfragments are Fab fragments wherein either the variable regions or theconstant regions of the heavy and light chain are exchanged. Bispecificantibody formats comprising crossover Fab fragments have been described,for example, in WO2009080252, WO2009080253, WO2009080251, WO2009080254,WO2010/136172, WO2010/145792 and WO2013/026831. In a particularembodiment, the first antigen binding moiety is a crossover Fab moleculewherein either the variable or the constant regions of the Fab lightchain and the Fab heavy chain are exchanged. Such modification preventmispairing of heavy and light chains from different Fab molecules,thereby improving the yield and purity of the T cell activatingbispecific antigen binding molecule of the invention in recombinantproduction. In a particular crossover Fab molecule useful for the T cellactivating bispecific antigen binding molecule of the invention, thevariable regions of the Fab light chain and the Fab heavy chain areexchanged. In another crossover Fab molecule useful for the T cellactivating bispecific antigen binding molecule of the invention, theconstant regions of the Fab light chain and the Fab heavy chain areexchanged.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at leastone light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 65.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, theheavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59,the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQID NO:65.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 55 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 64.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 64 or variants thereofthat retain functionality.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 31, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55, and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 64.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

(iii) a third antigen binding moiety capable of specific binding toFolR1.

In one embodiment, the third antigen binding moiety is a conventionalFab molecule. In one embodiment, the third antigen binding moiety is acrossover Fab molecule.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at leastone light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 65.(iii) a third antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 56 and SEQ ID NO: 57 and at leastone light chain CDR selected from the group of SEQ ID NO: 59, SEQ ID NO:60, SEQ ID NO: 65.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 56, theheavy chain CDR3 of SEQ ID NO:57, the light chain CDR1 of SEQ ID NO: 59,the light chain CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQID NO:65.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 55 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 64.(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 55 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 64.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at leastone light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, theheavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52,the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQID NO:54.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule. In one embodiment, the second antigen binding moiety is acrossover Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 49 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 51.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule. In one embodiment, the second antigen binding moiety is acrossover Fab molecule.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 51 or variants thereofthat retain functionality.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 31, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49, and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 51.

In one embodiment the T cell activating bispecific antigen bindingmolecule additionally comprises

(iii) a third antigen binding moiety capable of specific binding toFolR1.

In one embodiment, the third antigen binding moiety is a conventionalFab molecule. In one embodiment, the second antigen binding moiety is acrossover Fab molecule.

In one such embodiment the second and third antigen binding moietycapable of specific binding to FolR1 comprise identical heavy chaincomplementarity determining region (CDR) and light chain CDR sequences.In one such embodiment the third antigen binding moiety is identical tothe second antigen binding moiety.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 and atleast one light chain CDR selected from the group of SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34;(ii) a second antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 49 and at leastone light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54.(iii) a third antigen binding moiety capable of specific binding toFolate Receptor 1 (FolR1) comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 50 and at leastone light chain CDR selected from the group of SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54.

In one such embodiment the CD3 antigen binding moiety comprises theheavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO:38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ IDNO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3of SEQ ID NO:34 and the FolR1 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, theheavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52,the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQID NO:54.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

In one embodiment the T cell activating bispecific antigen bindingmolecule comprises

(i) a first antigen binding moiety which is a crossover Fab moleculecapable of specific binding to CD3 comprising a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 36 and a variable lightchain comprising an amino acid sequence of SEQ ID NO: 31.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 49 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 51.(iii) a third antigen binding moiety which is a Fab molecule capable ofspecific binding to Folate Receptor 1 (FolR1) comprising a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 49 and avariable light chain comprising an amino acid sequence of SEQ ID NO: 51.

In one embodiment, the second antigen binding moiety and the thirdantigen binding moiety are both a conventional Fab molecule.

Thus, in one embodiment, the invention relates to bispecific moleculeswherein two binding moieties confer specific binding to FolR1 and onebinding moiety confers specificity to the T cell activating antigen CD3.One of the heavy chains is modified to ensure proper pairing of theheavy and light chains, thus eliminating the need for a common lightchain approach. The presence of two FolR1 binding sites enablesappropriate engagement with the target antigen FolR1 and the activationof T cells.

The components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1A-I and are furtherdescribed below.

In some embodiments, said T cell activating bispecific antigen bindingmolecule further comprises an Fc domain composed of a first and a secondsubunit capable of stable association. Below exemplary embodiments of Tcell activating bispecific antigen binding molecule comprising an Fcdomain are described.

T Cell Activating Bispecific Antigen Binding Molecule Formats

As depicted above and in FIGS. 1A-I, in one embodiment the T cellactivating bispecific antigen binding molecules comprise at least twoFab fragments having identical light chains (VLCL) and having differentheavy chains (VHCL) which confer the specificities to two differentantigens, i.e. one Fab fragment is capable of specific binding to a Tcell activating antigen CD3 and the other Fab fragment is capable ofspecific binding to the target cell antigen FolR1.

In another embodiment the T cell activating bispecific antigen bindingmolecule comprises at least two antigen binding moieties (Fabmolecules), one of which is a crossover Fab molecule and one of which isa conventional Fab molecule. In one such embodiment the first antigenbinding moiety capable of specific binding to CD3 is a crossover Fabmolecule and the second antigen binding moiety capable of specificbinding to FolR is a conventional Fab molecule.

These components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIG. 1A-I.

In some embodiments, the first and second antigen binding moiety areeach fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe first or the second subunit of the Fc domain. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of a first and a second antigen binding moiety, anFc domain composed of a first and a second subunit, and optionally oneor more peptide linkers, wherein the first and second antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of the first or the second subunit of the Fc domain. In onesuch embodiment the first and second antigen binding moiety both are Fabfragments and have identical light chains (VLCL). In another suchembodiment the first antigen binding moiety capable of specific bindingto CD3 is a crossover Fab molecule and the second antigen binding moietycapable of specific binding to FolR is a conventional Fab molecule.

In one embodiment, the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain and the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of a first and a second antigen binding moiety, anFc domain composed of a first and a second subunit, and optionally oneor more peptide linkers, wherein the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding moiety, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or the second subunit of the Fc domain.In one such embodiment the first and second antigen binding moiety bothare Fab fragments and have identical light chains (VLCL). In anothersuch embodiment the first antigen binding moiety capable of specificbinding to CD3 is a crossover Fab molecule and the second antigenbinding moiety capable of specific binding to FolR is a conventional Fabmolecule. Optionally, the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the second antigen binding moiety mayadditionally be fused to each other.

In other embodiments, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a particular such embodiment, thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety. In a specific such embodiment, the T cellactivating bispecific antigen binding molecule essentially consists of afirst and a second antigen binding moiety, an Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second antigen binding moiety is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding moiety, and the first antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst or the second subunit of the Fc domain. In one such embodiment thefirst and second antigen binding moiety both are Fab fragments and haveidentical light chains (VLCL). In another such embodiment the firstantigen binding moiety capable of specific binding to CD3 is a crossoverFab molecule and the second antigen binding moiety capable of specificbinding to FolR is a conventional Fab molecule. Optionally, the Fablight chain of the first antigen binding moiety and the Fab light chainof the second antigen binding moiety may additionally be fused to eachother.

The antigen binding moieties may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n) (SEQ ID NO: 387), (SG₄)_(n) (SEQID NO: 388), (G₄S)_(n) (SEQ ID NO: 387) or G₄(SG₄)_(n) (SEQ ID NO: 389)peptide linkers. “n” is generally a number between 1 and 10, typicallybetween 2 and 4. A particularly suitable peptide linker for fusing theFab light chains of the first and the second antigen binding moiety toeach other is (G₄S)₂ (SEQ ID NO: 386). An exemplary peptide linkersuitable for connecting the Fab heavy chains of the first and the secondantigen binding moiety is EPKSC(D)-(G₄S)₂ (SEQ ID NOS 390 and 391).Additionally, linkers may comprise (a portion of) an immunoglobulinhinge region. Particularly where an antigen binding moiety is fused tothe N-terminus of an Fc domain subunit, it may be fused via animmunoglobulin hinge region or a portion thereof, with or without anadditional peptide linker.

It has been found by the inventors of the present invention that T cellactivating bispecific antigen binding molecule comprising two bindingmoieties specific for the target cell antigen FolR have superiorcharacteristics compared to T cell activating bispecific antigen bindingmolecule comprising only one binding moiety specific for the target cellantigen FolR.

Accordingly, in certain embodiments, the T cell activating bispecificantigen binding molecule of the invention further comprises a thirdantigen binding moiety which is a Fab molecule capable of specificbinding to FolR. In one such embodiment the second and third antigenbinding moiety capable of specific binding to FolR1 comprise identicalheavy chain complementarity determining region (CDR) and light chain CDRsequences, i.e., the heavy chain CDR sequences of the second antigenbinding moiety are the same as the heavy chain CDR sequences of thethird antigen binding moiety, and the light chain CDR sequences of thesecond antigen binding moiety are the same as the light chain CDRsequences of the third antigen binding moiety. In one such embodimentthe third antigen binding moiety is identical to the second antigenbinding moiety (i.e. they comprise the same amino acid sequences).

In one embodiment, the first and second antigen binding moiety are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst or second subunit of the Fc domain and the third antigen bindingmoiety is fused at the C-terminus of the Fab heavy chain to theN-terminus to the N-terminus of the Fab heavy chain of the first antigenbinding moiety. In a specific such embodiment, the T cell activatingbispecific antigen binding molecule essentially consists of a first, asecond and a third antigen binding moiety, an Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first and second antigen binding moiety are each fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain and the third antigen binding moiety is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first antigen binding moiety. In one such embodimentthe first, second and third antigen binding moiety are conventional Fabfragments and have identical light chains (VLCL). In another suchembodiment the first antigen binding moiety capable of specific bindingto CD3 is a crossover Fab molecule and the second and third antigenbinding moiety capable of specific binding to FolR is a conventional Fabmolecule. Optionally, the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the third antigen binding moiety mayadditionally be fused to each other.

In another aspect, the invention provides for a bispecific antibodycomprising a) a first antigen-binding site that competes for binding tohuman FolR1 with a reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 49 and a variable light chain domain of SEQ IDNO: 51; and b) a second antigen-binding site that competes for bindingto human CD3 with a reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ IDNO: 31, wherein binding competition is measured using a surface plasmonresonance assay.

In another aspect, the invention provides for a T cell activatingbispecific antigen binding molecule comprising a first antigen bindingmoiety capable of specific binding to CD3, and a second antigen bindingmoiety capable of specific binding to Folate Receptor 1 (FolR1), whereinthe T cell activating bispecific antigen binding molecule binds to thesame epitope on human FolR1 as a first reference antibody comprising avariable heavy chain domain (VH) of SEQ ID NO: 49 and a variable lightchain domain of SEQ ID NO: 51; and wherein the T cell activatingbispecific antigen binding molecule binds to the same epitope on humanCD3 as a second reference antibody comprising a variable heavy chaindomain (VH) of SEQ ID NO: 36 and a variable light chain domain of SEQ IDNO: 31.

In another aspect, the invention provides for a T cell activatingbispecific antigen binding molecule that comprises a first, second,third, fourth and fifth polypeptide chain that form a first, a secondand a third antigen binding moiety wherein the first antigen bindingmoiety is capable of binding CD3 and the second and the third antigenbinding moiety each are capable of binding Folate Receptor 1 (FolR1).The first and the second polypeptide chain comprise, in amino(N)-terminal to carboxyl (C)-terminal direction, a first light chainvariable domain (VLD1) and a first light chain constant domain (CLD1).The third polypeptide chain comprises, in N-terminal to C-terminaldirection, second light chain variable domain (VLD2) and a second heavychain constant domain 1 (CH1D2). The fourth polypeptide chain comprises,in N-terminal to C-terminal direction, a first heavy chain variabledomain (VHD1), a first heavy chain constant domain 1 (CH1D1), a firstheavy chain constant domain 2 (CH2D1) and a first heavy chain constantdomain 3 (CH3D1). The fifth polypeptide chain comprises VHD1, CH1D1, asecond heavy chain variable domain (VHD2), a second light chain constantdomain (CLD2), a second heavy chain constant domain 2 (CH2D2) and asecond heavy chain constant domain 3 (CH3D2). The third polypeptidechain and VHD2 and CLD2 of the fifth polypeptide chain form the firstantigen binding moiety capable of binding CD3. The second polypeptidechain and VHD1 and CH1D1 of the fifth polypeptide chain form the thirdbinding moiety capable of binding to FolR1. The first polypeptide chainand VHD1 and CH1D1 of the fourth polypeptide chain form the secondbinding moiety capable of binding to FolR1.

In another embodiment, the second and the third antigen binding moietyare each fused at the C-terminus of the Fab heavy chain to theN-terminus of the first or second subunit of the Fc domain, and thefirst antigen binding moiety is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding moiety. In a specific such embodiment, the T cell activatingbispecific antigen binding molecule essentially consists of a first, asecond and a third antigen binding moiety, an Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second and third antigen binding moiety are each fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain and the first antigen binding moiety is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the third antigen binding moiety. In one such embodimentthe first, second and third antigen binding moiety are conventional Fabfragments and have identical light chains (VLCL). In another suchembodiment the first antigen binding moiety capable of specific bindingto CD3 is a crossover Fab molecule and the second and third antigenbinding moiety capable of specific binding to FolR is a conventional Fabmolecule. Optionally, the Fab light chain of the first antigen bindingmoiety and the Fab light chain of the second antigen binding moiety mayadditionally be fused to each other.

The antigen binding moieties may be fused to the Fc domain directly orthrough a peptide linker. In a particular embodiment the antigen bindingmoieties are each fused to the Fc domain through an immunoglobulin hingeregion. In a specific embodiment, the immunoglobulin hinge region is ahuman IgG₁ hinge region.

In one embodiment the first and the second antigen binding moiety andthe Fc domain are part of an immunoglobulin molecule. In a particularembodiment the immunoglobulin molecule is an IgG class immunoglobulin.In an even more particular embodiment the immunoglobulin is an IgG₁subclass immunoglobulin. In another embodiment the immunoglobulin is anIgG₄ subclass immunoglobulin. In a further particular embodiment theimmunoglobulin is a human immunoglobulin. In other embodiments theimmunoglobulin is a chimeric immunoglobulin or a humanizedimmunoglobulin.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moiety,wherein the first, second and third antigen binding moiety areconventional Fab fragments and have identical light chains (VLCL),wherein the first antigen binding moiety capable of specific binding toCD3 comprises at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 37, SEQ IDNO: 38 and SEQ ID NO: 39 and at least one light chain CDR selected fromthe group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and thesecond and the third antigen binding moiety capable of specific bindingto FolR1 comprise at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 16, SEQ IDNO: 17 and SEQ ID NO: 18 and at least one light chain CDR selected fromthe group of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moiety,wherein the first, second and third antigen binding moiety areconventional Fab fragments and have identical light chains (VLCL),wherein the first antigen binding moiety capable of specific binding toCD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO:36, a variable light chain comprising a sequence of SEQ ID NO: 31; andthe second and the third antigen binding moiety capable of specificbinding to FolR1 comprise a variable heavy chain comprising a sequenceof SEQ ID NO: 15, a variable light chain comprising a sequence of SEQ IDNO: 31.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moietyand the first antigen binding moiety capable of specific binding to CD3is a crossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged,comprising at least one heavy chain complementarity determining region(CDR) selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38and SEQ ID NO: 39 and at least one light chain CDR selected from thegroup of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; and the secondand the third antigen binding moiety capable of specific binding toFolR1 comprise at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 8, SEQ IDNO: 56 and SEQ ID NO: 57 and at least one light chain CDR selected fromthe group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 65.

In a particular embodiment said T cell activating bispecific antigenbinding molecule the first and the second antigen binding moiety and theFc domain are part of an immunoglobulin molecule, and the third antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moietyand the first antigen binding moiety capable of specific binding to CD3is a crossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged,wherein the first antigen binding moiety capable of specific binding toCD3 comprises a variable heavy chain comprising a sequence of SEQ ID NO:36, a variable light chain comprising a sequence of SEQ ID NO: 31; andthe second and the third antigen binding moiety capable of specificbinding to FolR1 comprise a variable heavy chain comprising a sequenceof SEQ ID NO: 55, a variable light chain comprising a sequence of SEQ IDNO: 65.

In one embodiment the T cell activating bispecific antigen bindingmolecule is monovalent for each antigen. In a particular embodiment theT cell activating bispecific antigen binding molecule can bind to humanCD3 and human folate receptor alpha (FolR1) and was made withoutemploying a hetero-dimerization approach, such as, e.g., knob-into-holetechnology. For example, the molecule can be produced by employing acommon light chain library and CrossMab technology. In a particularembodiment, The variable region of the CD3 binder is fused to the CH1domain of a standard human IgG1 antibody to form the VLVH crossedmolecule (fused to Fc) which is common for both specificities. Togenerate the crossed counterparts (VHCL), a CD3 specific variable heavychain domain is fused to a constant human λ light chain whereas avariable heavy chain domain specific for human FolR1 (e.g., isolatedfrom a common light chain library) is fused to a constant human a lightchain. The resulting desired molecule with correctly paired chainscomprises both kappa and lambda light chains or fragments thereof.Consequently, this desired bispecific molecule species can be purifiedfrom mispaired or homodimeric species with sequential purification stepsselecting for kappa and lambda light chain, in either sequence. In oneparticular embodiment, purification of the desired bispecific antibodyemploys subsequent purification steps with KappaSelect andLambdaFabSelect columns (GE Healthcare) to remove undesired homodimericantibodies.

Fc Domain

The Fc domain of the T cell activating bispecific antigen bindingmolecule consists of a pair of polypeptide chains comprising heavy chaindomains of an immunoglobulin molecule. For example, the Fc domain of animmunoglobulin G (IgG) molecule is a dimer, each subunit of whichcomprises the CH2 and CH3 IgG heavy chain constant domains. The twosubunits of the Fc domain are capable of stable association with eachother. In one embodiment the T cell activating bispecific antigenbinding molecule of the invention comprises not more than one Fc domain.

In one embodiment according the invention the Fc domain of the T cellactivating bispecific antigen binding molecule is an IgG Fc domain. In aparticular embodiment the Fc domain is an IgG₁ Fc domain. In anotherembodiment the Fc domain is an IgG₄ Fc domain. In a more specificembodiment, the Fc domain is an IgG₄ Fc domain comprising an amino acidsubstitution at position S228 (Kabat numbering), particularly the aminoacid substitution S228P. This amino acid substitution reduces in vivoFab arm exchange of IgG₄ antibodies (see Stubenrauch et al., DrugMetabolism and Disposition 38, 84-91 (2010)). In a further particularembodiment the Fc domain is human.

Fc Domain Modifications Promoting Heterodimerization

T cell activating bispecific antigen binding molecules according to theinvention comprise different antigen binding moieties, fused to one orthe other of the two subunits of the Fc domain, thus the two subunits ofthe Fc domain are typically comprised in two non-identical polypeptidechains. Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of T cell activatingbispecific antigen binding molecules in recombinant production, it willthus be advantageous to introduce in the Fc domain of the T cellactivating bispecific antigen binding molecule a modification promotingthe association of the desired polypeptides.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecule according to theinvention comprises a modification promoting the association of thefirst and the second subunit of the Fc domain. The site of mostextensive protein-protein interaction between the two subunits of ahuman IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in oneembodiment said modification is in the CH3 domain of the Fc domain.

In a specific embodiment said modification is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the T cell activating bispecific antigenbinding molecule an amino acid residue is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable.

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain the threonine residue at position 366 is replaced with atryptophan residue (T366W), and in the CH3 domain of the second subunitof the Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V). In one embodiment, in the second subunit of theFc domain additionally the threonine residue at position 366 is replacedwith a serine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, thus further stabilizing the dimer (Carter, J ImmunolMethods 248, 7-15 (2001)).

In a particular embodiment the antigen binding moiety capable of bindingto CD3 is fused (optionally via the antigen binding moiety capable ofbinding to a target cell antigen) to the first subunit of the Fc domain(comprising the “knob” modification). Without wishing to be bound bytheory, fusion of the antigen binding moiety capable of binding to CD3to the knob-containing subunit of the Fc domain will (further) minimizethe generation of antigen binding molecules comprising two antigenbinding moieties capable of binding to CD3 (steric clash of twoknob-containing polypeptides).

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Fc Domain Modifications Abolishing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the T cell activating bispecific antigenbinding molecule favorable pharmacokinetic properties, including a longserum half-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. At the same timeit may, however, lead to undesirable targeting of the T cell activatingbispecific antigen binding molecule to cells expressing Fc receptorsrather than to the preferred antigen-bearing cells. Moreover, theco-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the T cell activating properties andthe long half-life of the antigen binding molecule, results in excessiveactivation of cytokine receptors and severe side effects upon systemicadministration. Activation of (Fc receptor-bearing) immune cells otherthan T cells may even reduce efficacy of the T cell activatingbispecific antigen binding molecule due to the potential destruction ofT cells e.g. by NK cells.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecules according to theinvention exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. Inone such embodiment the Fc domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG₁ Fc domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG₁ Fc domain domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain). In oneembodiment, the Fc domain domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) does notsubstantially bind to an Fc receptor and/or induce effector function. Ina particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof CDC, ADCC, ADCP, and cytokine secretion. In a particular embodimentthe effector function is ADCC. In one embodiment the Fc domain domainexhibits substantially similar binding affinity to neonatal Fc receptor(FcRn), as compared to a native IgG₁ Fc domain domain. Substantiallysimilar binding to FcRn is achieved when the Fc domain (or the T cellactivating bispecific antigen binding molecule comprising said Fcdomain) exhibits greater than about 70%, particularly greater than about80%, more particularly greater than about 90% of the binding affinity ofa native IgG₁ Fc domain (or the T cell activating bispecific antigenbinding molecule comprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the T cell activating bispecific antigen binding moleculecomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one embodiment the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In one embodiment the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In embodiments where there is more than oneamino acid mutation that reduces the binding affinity of the Fc domainto the Fc receptor, the combination of these amino acid mutations mayreduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the T cell activating bispecific antigen binding moleculecomprising an engineered Fc domain exhibits less than 20%, particularlyless than 10%, more particularly less than 5% of the binding affinity toan Fc receptor as compared to a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain. In a particularembodiment the Fc receptor is an Fcγ receptor. In some embodiments theFc receptor is a human Fc receptor. In some embodiments the Fc receptoris an activating Fc receptor. In a specific embodiment the Fc receptoris an activating human Fcγ receptor, more specifically human FcγRIIIa,FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, bindingto each of these receptors is reduced. In some embodiments bindingaffinity to a complement component, specifically binding affinity toC1q, is also reduced. In one embodiment binding affinity to neonatal Fcreceptor (FcRn) is not reduced. Substantially similar binding to FcRn,i.e. preservation of the binding affinity of the Fc domain to saidreceptor, is achieved when the Fc domain (or the T cell activatingbispecific antigen binding molecule comprising said Fc domain) exhibitsgreater than about 70% of the binding affinity of a non-engineered formof the Fc domain (or the T cell activating bispecific antigen bindingmolecule comprising said non-engineered form of the Fc domain) to FcRn.The Fc domain, or T cell activating bispecific antigen binding moleculesof the invention comprising said Fc domain, may exhibit greater thanabout 80% and even greater than about 90% of such affinity. In certainembodiments the Fc domain of the T cell activating bispecific antigenbinding molecule is engineered to have reduced effector function, ascompared to a non-engineered Fc domain. The reduced effector functioncan include, but is not limited to, one or more of the following:reduced complement dependent cytotoxicity (CDC), reducedantibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329. In a more specific embodiment the Fcdomain comprises an amino acid substitution at a position selected fromthe group of L234, L235 and P329. In some embodiments the Fc domaincomprises the amino acid substitutions L234A and L235A. In one suchembodiment, the Fc domain is an IgG₁ Fc domain, particularly a humanIgG₁ Fc domain. In one embodiment the Fc domain comprises an amino acidsubstitution at position P329. In a more specific embodiment the aminoacid substitution is P329A or P329G, particularly P329G. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329 and a further amino acid substitution at a positionselected from E233, L234, L235, N297 and P331. In a more specificembodiment the further amino acid substitution is E233P, L234A, L235A,L235E, N297A, N297D or P331S. In particular embodiments the Fc domaincomprises amino acid substitutions at positions P329, L234 and L235. Inmore particular embodiments the Fc domain comprises the amino acidmutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment,the Fc domain is an IgG₁ Fc domain, particularly a human IgG₁Fc domain.The “P329G LALA” combination of amino acid substitutions almostcompletely abolishes Fcγ receptor binding of a human IgG₁ Fc domain, asdescribed in PCT publication no. WO 2012/130831, incorporated herein byreference in its entirety. WO 2012/130831 also describes methods ofpreparing such mutant Fc domains and methods for determining itsproperties such as Fc receptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain of the T cell activating bispecificantigen binding molecules of the invention is an IgG₄ Fc domain,particularly a human IgG₄ Fc domain. In one embodiment the IgG₄ Fcdomain comprises amino acid substitutions at position S228, specificallythe amino acid substitution S228P. To further reduce its bindingaffinity to an Fc receptor and/or its effector function, in oneembodiment the IgG₄ Fc domain comprises an amino acid substitution atposition L235, specifically the amino acid substitution L235E. Inanother embodiment, the IgG₄ Fc domain comprises an amino acidsubstitution at position P329, specifically the amino acid substitutionP329G. In a particular embodiment, the IgG₄ Fc domain comprises aminoacid substitutions at positions S228, L235 and P329, specifically aminoacid substitutions S228P, L235E and P329G. Such IgG₄ Fc domain mutantsand their Fcγ receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety.

In a particular embodiment the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G.

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing. Binding toFc receptors can be easily determined e.g. by ELISA, or by SurfacePlasmon Resonance (SPR) using standard instrumentation such as a BIAcoreinstrument (GE Healthcare), and Fc receptors such as may be obtained byrecombinant expression. A suitable such binding assay is describedherein. Alternatively, binding affinity of Fc domains or cell activatingbispecific antigen binding molecules comprising an Fc domain for Fcreceptors may be evaluated using cell lines known to express particularFc receptors, such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or a T cell activating bispecificantigen binding molecule comprising an Fc domain, can be measured bymethods known in the art. A suitable assay for measuring ADCC isdescribed herein. Other examples of in vitro assays to assess ADCCactivity of a molecule of interest are described in U.S. Pat. No.5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986)and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in a animal model such as that disclosed in Clynes et al.,Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the T cell activating bispecificantigen binding molecule is able to bind C1q and hence has CDC activity.See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

Fc Domain Modifications Promoting Heterodimerization

The T cell activating bispecific antigen binding molecule of theinvention comprise different antigen binding moieties, some of which arefused to one or the other of the two subunits of the Fc domain, thus thetwo subunits of the Fc domain are typically comprised in twonon-identical polypeptide chains. Recombinant co-expression of thesepolypeptides and subsequent dimerization leads to several possiblecombinations of the two polypeptides. To improve the yield and purity ofthe bispecific antibodies of the invention in recombinant production, itwill thus be advantageous to introduce in the Fc domain of thebispecific antibodies of the invention a modification promoting theassociation of the desired polypeptides.

Accordingly, in particular embodiments, the Fc domain of the bispecificantibodies of the invention comprises a modification promoting theassociation of the first and the second subunit of the Fc domain. Thesite of most extensive protein-protein interaction between the twosubunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.Thus, in one embodiment said modification is in the CH3 domain of the Fcdomain.

In a specific embodiment, said modification is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain. The knob-into-holetechnology is described e.g. in U.S. Pat. Nos. 5,731,168; 7,695,936;Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth248, 7-15 (2001).

Generally, the method involves introducing a protuberance (“knob”) atthe interface of a first polypeptide and a corresponding cavity (“hole”)in the interface of a second polypeptide, such that the protuberance canbe positioned in the cavity so as to promote heterodimer formation andhinder homodimer formation. Protuberances are constructed by replacingsmall amino acid side chains from the interface of the first polypeptidewith larger side chains (e.g. tyrosine or tryptophan). Compensatorycavities of identical or similar size to the protuberances are createdin the interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the bispecific antibodies of the inventionan amino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theCH3 domain of the first subunit which is positionable in a cavity withinthe CH3 domain of the second subunit, and in the CH3 domain of thesecond subunit of the Fc domain an amino acid residue is replaced withan amino acid residue having a smaller side chain volume, therebygenerating a cavity within the CH3 domain of the second subunit withinwhich the protuberance within the CH3 domain of the first subunit ispositionable.

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain the threonine residue at position 366 is replaced with atryptophan residue (T366W), and in the CH3 domain of the second subunitof the Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V). In one embodiment, in the second subunit of theFc domain additionally the threonine residue at position 366 is replacedwith a serine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter, J Immunol Methods248, 7-15 (2001)). In an alternative embodiment a modification promotingassociation of the first and the second subunit of the Fc domaincomprises a modification mediating electrostatic steering effects, e.g.as described in WO 2009/089004. Generally, this method involvesreplacement of one or more amino acid residues at the interface of thetwo Fc domain subunits by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

In one embodiment, a T cell activating bispecific antigen bindingmolecule that binds to FolR1 and CD3 according to any of the aboveembodiments comprises an Immunoglobulin G (IgG) molecule with twobinding sites specific for FolR1, wherein the Fc part of the first heavychain comprises a first dimerization module and the Fc part of thesecond heavy chain comprises a second dimerization module allowing aheterodimerization of the two heavy chains of the IgG molecule.

In a further preferred embodiment, the first dimerization modulecomprises knobs and the second dimerization module comprises holesaccording to the knobs into holes strategy (see Carter P.; Ridgway J. B.B.; Presta L. G.: Immunotechnology, Volume 2, Number 1, February 1996,pp. 73-73(1)).

Biological Properties and Functional Characteristics of T CellActivating Bispecific Antigen Binding Molecules

One of skill in the art can appreciate the advantageous efficiency of amolecule that selectively distinguishes between cancerous andnon-cancerous, healthy cells. One way to accomplish this goal is byappropriate target selection. Markers expressed exclusively on tumorcells can be employed to selectively target effector molecules or cellsto tumor cells while sparing normal cells that do not express suchmarker. However, in some instances, so called tumor cell markers arealso expressed in normal tissue, albeit at lower levels. This expressionin normal tissue raises the possibility of toxicity. Thus, there was aneed in the art for molecules that can more selectively target tumorcells. The invention described herein provides for T cell activatingbispecific antigen binding molecules that selectively targetFolR1-positive tumor cells and not normal, non-cancerous cells thatexpress FolR1 at low levels or not at all. In one embodiment, the T cellactivating bispecific antigen binding molecule comprises at least two,preferably two, FolR1 binding moieties of relatively low affinity thatconfer an avidity effect which allows for differentiation between highand low FolR1 expressing cells. Because tumor cells express FolR1 athigh or intermediate levels, this embodiment of the inventionselectively binds to, and/or induces killing of, tumor cells and notnormal, non-cancerous cells that express FolR1 at low levels or not atall. In one embodiment, the T cell activating bispecific antigen bindingmolecule is in the 2+1 inverted format. In one embodiment, the T cellactivating bispecific antigen binding molecule induces T cell mediatedkilling of FolR1-positive tumor cells and not non-tumor cells andcomprises a CD3 antigen binding moiety that comprises the heavy chainCDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavychain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32, thelight chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ IDNO:34 and two FolR1 antigen binding moieties that each comprise theheavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9,the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO:52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 ofSEQ ID NO:54.

In one specific embodiment, the T cell activating bispecific antigenbinding molecule does not induce killing of a normal cells having lessthan about 1000 copies of FolR1 its surface. In addition to the aboveadvantageous characteristics, one embodiment of the invention does notrequire chemical cross linking or a hybrid approach to be produced.Accordingly, in one embodiment, the invention provides for T cellactivating bispecific antigen binding molecule capable of production inCHO cells. In one embodiment, the T cell activating bispecific antigenbinding molecule comprises humanized and human polypeptides. In oneembodiment, the T cell activating bispecific antigen binding moleculedoes not cause FcgR crosslinking. In one such embodiment, the T cellactivating bispecific antigen binding molecule is capable of productionin CHO cells and comprises a CD3 antigen binding moiety that comprisesthe heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ IDNO: 38, the heavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 ofSEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the lightchain CDR3 of SEQ ID NO:34 and two FolR1 antigen binding moieties thateach comprise the heavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50, the light chainCDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ ID NO: 53, and thelight chain CDR3 of SEQ ID NO:54.

As noted above, some embodiments contemplated herein include T cellactivating bispecific antigen binding molecules having two bindingmoieties that confer specific binding to FolR1 and one binding moietythat confers specificity to the T cell activating antigen CD3, whereineach individual FolR1 binding moiety engages the antigen with lowaffinity. Because the molecule comprises two antigen binding moietiesthat confer binding to FolR1, the overall avidity of the molecule,nevertheless, provides effective binding to FolR1-expressing targetcells and activation of T cells to induce T cell effector function.Considering that while FolR1 is expressed at various level on tumorcells, it is also expressed at very low levels (e.g., less than about1000 copies on the cell surface) in certain normal cells, one of skillin the art can readily recognize the advantageous efficiency of such amolecule for use as a therapeutic agent. Such molecule selectivelytargets tumor cells over normal cells. Such molecule, thus, can beadministered to an individual in need thereof with significantly lessconcern about toxicity resulting from FolR1 positive normal cellscompared to molecules that bind to FolR1 with high affinity to induceeffector function.

In one embodiment, the T cell activating bispecific antigen bindingmolecule binds human FolR1 with an apparent K_(D) of about 5.36 pM toabout 4 nM. In one embodiment, the T cell activating bispecific antigenbinding molecule binds human and cynomolgus FolR1 with an apparent K_(D)of about 4 nM. In one embodiment, the T cell activating bispecificantigen binding molecule binds murine FolR1 with an apparent K_(D) ofabout 1.5 nM. In one embodiment, the T cell activating bispecificantigen binding molecule binds human FolR1 with a monovalent bindingK_(D) of at least about 1000 nM. In a specific embodiment, the T cellactivating bispecific antigen binding molecule binds human andcynomolgus FolR1 with an apparent K_(D) of about 4 nM, binds murineFolR1 with an apparent K_(D) of about 1.5 nM, and comprises a CD3antigen binding moiety that comprises the heavy chain CDR1 of SEQ ID NO:37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQID NO:39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 ofSEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO:34 and two FolR1antigen binding moieties that each comprise the heavy chain CDR1 of SEQID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 ofSEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 52, the light chainCDR2 of SEQ ID NO: 53, and the light chain CDR3 of SEQ ID NO:54. In oneembodiment, the T cell activating bispecific antigen binding moleculebinds human FolR1 with a monovalent binding K_(D) of at least about 1000nM and comprises a CD3 antigen binding moiety that comprises the heavychain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, theheavy chain CDR3 of SEQ ID NO:39, the light chain CDR1 of SEQ ID NO: 32,the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQID NO:34 and two FolR1 antigen binding moieties that each comprise theheavy chain CDR1 of SEQ ID NO: 8, the heavy chain CDR2 of SEQ ID NO: 9,the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO:52, the light chain CDR2 of SEQ ID NO: 53, and the light chain CDR3 ofSEQ ID NO:54. As described above, the T cell activating bispecificantigen binding molecules contemplated herein can induce T cell effectorfunction, e.g., cell surface marker expression, cytokine production, Tcell mediated killing. In one embodiment, the T cell activatingbispecific antigen binding molecule induces T cell mediated killing ofthe FolR1-expressing target cell, such as a human tumor cell, in vitro.In one embodiment, the T cell is a CD8 positive T cell. Examples ofFolR1-expressing human tumor cells include but are not limited to Hela,Skov-3, HT-29, and HRCEpiC cells. Other FolR1 positive human cancercells that can be used for in vitro testing are readily available to theskilled artisan. In one embodiment, the T cell activating bispecificantigen binding molecule induces T cell mediated killing of theFolR1-expressing human tumor cell in vitro with an EC50 of between about36 pM and about 39573 pM after 24 hours. Specifically contemplated are Tcell activating bispecific antigen binding molecules that induce T cellmediated killing of the FolR1-expressing tumor cell in vitro with anEC50 of about 36 pM after 24 hours. In one embodiment, the T cellactivating bispecific antigen binding molecule induces T cell mediatedkilling of the FolR1-expressing tumor cell in vitro with an EC50 ofabout 178.4 pM after 24 hours. In one embodiment, the T cell activatingbispecific antigen binding molecule induces T cell mediated killing ofthe FolR1-expressing tumor cell in vitro with an EC50 of about 134.5 pMor greater after 48 hours. The EC50 can be measure by methods known inthe art, for example by methods disclosed herein by the examples.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of any of the above embodiments induces upregulation of cellsurface expression of at least one of CD25 and CD69 on the T cell asmeasured by flow cytometry. In one embodiment, the T cell is a CD4positive T cell or a CD8 positive T cell.

In one embodiment, the T cell activating bispecific antigen bindingmolecule of any of the above embodiments binds to FolR1 expressed on ahuman tumor cell. In one embodiment, the T cell activating bispecificantigen binding molecule of any of the above embodiments binds to aconformational epitope on human FolR1. In one embodiment, the T cellactivating bispecific antigen binding molecule of any of the aboveembodiments does not bind to human Folate Receptor 2 (FolR2) or to humanFolate Receptor 3 (FolR3). In one embodiment of the T cell activatingbispecific antigen binding molecule of any of the above embodiments, theantigen binding moiety binds to a FolR polypeptide comprising the aminoacids 25 to 234 of human FolR1 (SEQ ID NO:227). In one embodiment of theT cell activating bispecific antigen binding molecule of any of theabove embodiments, the FolR1 antigen binding moiety binds to a FolRpolypeptide comprising the amino acid sequence of SEQ ID NOs:227, 230and 231, and wherein the FolR antigen binding moiety does not bind to aFolR polypeptide comprising the amino acid sequence of SEQ ID NOs:228and 229. In one specific embodiment, the T cell activating bispecificantigen binding molecule comprises a FolR1 antigen binding moiety thatbinds to a FolR1 polypeptide comprising the amino acid sequence of SEQID NOs:227, 230 and 231, and wherein the FolR1 antigen binding moietydoes not bind to a FolR polypeptide comprising the amino acid sequenceof SEQ ID NOs:228 and 229, and comprises a CD3 antigen binding moietythat comprises the heavy chain CDR1 of SEQ ID NO: 37, the heavy chainCDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO:39, the lightchain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, andthe light chain CDR3 of SEQ ID NO:34 and two FolR1 antigen bindingmoieties that each comprise the heavy chain CDR1 of SEQ ID NO: 8, theheavy chain CDR2 of SEQ ID NO: 9, the heavy chain CDR3 of SEQ ID NO:50,the light chain CDR1 of SEQ ID NO: 52, the light chain CDR2 of SEQ IDNO: 53, and the light chain CDR3 of SEQ ID NO:54.

With respect to the FolR1, the T cell activating bispecific antigenbinding molecules contemplated herein can have agonist, antagonist orneutral effect. Examples of agonist effect include induction orenhancement of signaling through the FolR1 upon engagement by the FolR1binding moiety with the FolR1 receptor on the target cell. Examples ofantagonist activity include abrogation or reduction of signaling throughthe FolR1 upon engagement by the FolR1 binding moiety with the FolR1receptor on the target cell. This can, for example, occur by blocking orreducing the interaction between folate with FolR1.

Exemplary PD-1 Axis Binding Antagonists for Use in the Invention

Provided herein are methods for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a T cell activating bispecific antigen bindingmolecule and a PD-1 axis binding antagonist. For example, a PD-1 axisbinding antagonist includes a PD-1 binding antagonist, a PDL1 bindingantagonist and a PDL2 binding antagonist. Alternative names for “PD-1”include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1,B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc,and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1,PDL1 and PDL2.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect the PD-ligand binding partners are PDL1 and/or PDL2. Inanother embodiment, a PDL1 binding antagonist is a molecule thatinhibits the binding of PDL1 to its binding partners. In a specificaspect, PDL1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PDL2 binding antagonist is a molecule that inhibits thebinding of PDL2 to its binding partners. In a specific aspect, a PDL2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide. In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or achimeric antibody). In some embodiments, the anti-PD-1 antibody isselected from the group consisting of nivolumab, pembrolizumab, andCT-011. In some embodiments, the PD-1 binding antagonist is animmunoadhesin (e.g., an immunoadhesin comprising an extracellular orPD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g.,an Fc region of an immunoglobulin sequence). In some embodiments, thePD-1 binding antagonist is AMP-224. Nivolumab, also known asMDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is ananti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also knownas MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is ananti-PD-1 antibody described in WO2009/114335. CT-011, also known ashBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611.AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptordescribed in WO2010/027827 and WO2011/066342.

In some embodiments, the anti-PD-1 antibody is nivolumab (CAS RegistryNumber. 946414-94-4). In a still further embodiment, provided is anisolated anti-PD-1 antibody comprising a heavy chain variable regioncomprising the heavy chain variable region amino acid sequence from SEQID NO:274 and/or a light chain variable region comprising the lightchain variable region amino acid sequence from SEQ ID NO:275. In a stillfurther embodiment, provided is an isolated anti-PD-1 antibodycomprising a heavy chain and/or a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:

(SEQ ID NO: 274) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK,or(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 275) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

In some embodiments, the anti-PD-1 antibody is pembrolizumab (CASRegistry Number. 1374853-91-4). In a still further embodiment, providedis an isolated anti-PD-1 antibody comprising a heavy chain variableregion comprising the heavy chain variable region amino acid sequencefrom SEQ ID NO:276 and/or alight chain variable region comprising thelight chain variable region amino acid sequence from SEQ ID NO:277. In astill further embodiment, provided is an isolated anti-PD-1 antibodycomprising a heavy chain and/or alight chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:

(SEQ ID NO: 276) QVQLVQSGVE VKKPGASVKVSCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTLTTDSSTTTAY MELKSLQFDD TAVYYCARRDYRFDMGFDYW GQGTTVTVSSASTKGPSVFP LAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCP APEFLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS  LSLSLGK,or(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 277) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In some embodiments, the PDL1 binding antagonist is anti-PDL1 antibody.In some embodiments, the anti-PDL1 binding antagonist is selected fromthe group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736.MDX-1105, also known as BMS-936559, is an anti-PDL1 antibody describedin WO2007/005874. Antibody YW243.55.S70 (heavy and light chain variableregion sequences shown in SEQ ID Nos. 20 and 21, respectively) is ananti-PDL1 described in WO 2010/077634 A1. MEDI4736 is an anti-PDL1antibody described in WO2011/066389 and US2013/034559, each incorporatedherein by reference as if set forth in their entirety.

Examples of anti-PDL1 antibodies useful for the methods of thisinvention, and methods for making thereof are described in PCT patentapplication WO 2010/077634 A1 and U.S. Pat. No. 8,217,149, eachincorporated herein by reference as if set forth in their entirety.

In some embodiments, the PD-1 axis binding antagonist is an anti-PDL1antibody. In some embodiments, the anti-PDL1 antibody is capable ofinhibiting binding between PDL1 and PD-1 and/or between PDL1 and B7-1.In some embodiments, the anti-PDL1 antibody is a monoclonal antibody. Insome embodiments, the anti-PDL1 antibody is an antibody fragmentselected from the group consisting of Fab, Fab′-SH, Fv, scFv, and(Fab′)2 fragments. In some embodiments, the anti-PDL1 antibody is ahumanized antibody. In some embodiments, the anti-PDL1 antibody is ahuman antibody.

The anti-PDL1 antibodies useful in this invention, includingcompositions containing such antibodies, such as those described in WO2010/077634 A, may be used in combination with a T cell activatingantigen binding molecule, and, optionally an anti-TIM3 antagonistantibody, to treat cancer. In some embodiments, the anti-PDL1 antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:382 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:383.

In one embodiment, the anti-PDL1 antibody contains a heavy chainvariable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3sequence, wherein:

(a) the HVR-H1 sequence is (SEQ ID NO: 283) GFTFSX1SWIH;(b) the HVR-H2 sequence is (SEQ ID NO: 284) AWIX2PYGGSX3YYADSVKG;(c) the HVR-H3 sequence is (SEQ ID NO: 285) RHWPGGFDY;further wherein: X1 is D or G; X2 is S or L; X3 is T or S.

In one specific aspect, X1 is D; X2 is S and X3 is T. In another aspect,the polypeptide further comprises variable region heavy chain frameworksequences juxtaposed between the HVRs according to the formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVRH3)-(HC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the framework sequences are VHsubgroup III consensus framework. In a still further aspect, at leastone of the framework sequences is the following:

HC-FR1 is (SEQ ID NO: 295) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 is(SEQ ID NO: 296) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 297)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 is (SEQ ID NO: 298) WGQGTLVTVSA.

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain comprising an HVR-L1, HVR-L2and HVR-L3, wherein:

(a) the HVR-L1 sequence is (SEQ ID NO: 286) RASQX4X5X6TX7X8A;(b) the HVR-L2 sequence is (SEQ ID NO: 287) SASX9LX10S,;(c) the HVR-L3 sequence is (SEQ ID NO: 288) QQX11X12X13X14PX15T;further wherein: X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F;X8 is V or L; X9 is F or T; X10 is Y or A; X11 is Y, G, F, or S; X12 isL, Y, F or W; X13 is Y, N, A, T, G, F or I; X14 is H, V, P, T or I; X15is A, W, R, P or T.

In a still further aspect, X4 is D; X5 is V; X6 is S X7 is A; X8 is V;X9 is F; X10 is Y; X11 is Y; X12 is L; X13 is Y; X14 is H; X15 is A. Ina still further aspect, the light chain further comprises variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LCFR4).

In a still further aspect, the framework sequences are derived fromhuman consensus framework sequences. In a still further aspect, theframework sequences are VL kappa I consensus framework. In a stillfurther aspect, at least one of the framework sequence is the following:

LC-FR1 is (SEQ ID NO: 300) DIQMTQSPSSLSASVGDRVTITC LC-FR2 is(SEQ ID NO: 301) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 302)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 303) FGQGTKVEIKR.

In another embodiment, provided is an isolated anti-PDL1 antibody orantigen binding fragment comprising a heavy chain and a light chainvariable region sequence, wherein:

(a) the heavy chain comprises and HVR-H1, HVR-H2 and HVR-H3, whereinfurther:

(i) the HVR-H1 sequence is (SEQ ID NO: 283) GFTFSX1SWIH(ii) the HVR-H2 sequence is (SEQ ID NO: 284) AWIX2PYGGSX3YYADSVKG(iii) the HVR-H3 sequence is (SEQ ID NO: 285) RHWPGGFDY(b) the light chain comprises and HVR-L1, HVR-L2 and HVR-L3, whereinfurther:

(i) the HVR-L1 sequence is (SEQ ID NO: 286) RASQX4X5X6TX7X8A(ii) the HVR-L2 sequence is (SEQ ID NO: 287) SASX9LX10S(iii) the HVR-L3 sequence is (SEQ ID NO: 288) QQX11X12X13X14PX15T

Further wherein: X1 is D or G; X2 is S or L; X3 is T or S; X4 is D or V;X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T;X10 is Y or A; X11 is Y, G, F, or S; X12 is L, Y, F or W; X13 is Y, N,A, T, G, F or I; X14 is H, V, P, T or I; X15 is A, W, R, P or T.

In a specific aspect, X1 is D; X2 is S and X3 is T. In another aspect,X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y; X11 isY; X12 is L; X13 is Y; X14 is H; X15 is A. In yet another aspect, X1 isD; X2 is S and X3 is T, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9is F; X10 is Y; X11 is Y; X12 is L; X13 is Y; X14 is H and X15 is A.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HCFR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVRL2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences is thefollowing:

HC-FR1 (SEQ ID NO: 295) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2(SEQ ID NO: 296) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 297)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 298) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 300) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 301)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 302) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 303) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effectorless Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PDL1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

(a) the heavy chain further comprises and HVR-H1, HVR-H2 and an HVRH3sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ IDNO:289), AWISPYGGSTYYADSVKG (SEQ ID NO:290), and RHWPGGFDY (SEQ IDNO:291), respectively, or

(b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ IDNO:292), SASFLYS (SEQ ID NO:293) and QQYLYHPAT (SEQ ID NO:294),respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HCFR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup I, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences is the following:

HC-FR1 (SEQ ID NO: 295) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2(SEQ ID NO: 296) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 297)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 298) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 300) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 301)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 302) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 303) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effectorless Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In a still further embodiment, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 382) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR RHWPGGFDYWGQGTLVTVSA,or(b) the light chain sequence has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 383) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HCFR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework.

In a still further aspect, one or more of the heavy chain frameworksequences is the following:

HC-FR1 (SEQ ID NO: 295) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2(SEQ ID NO: 296) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 297)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 298) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 300) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 301)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 302) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 303) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In another further embodiment, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 280) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR RHWPGGFDYWGQGTLVTVSS,or(b) the light chain sequence has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 383) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In a still further embodiment, provided is an isolated anti-PDL1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 281) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK,or(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 282) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HCFR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework.

In a still further aspect, one or more of the heavy chain frameworksequences is the following:

HC-FR1 (SEQ ID NO: 295) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2(SEQ ID NO: 296) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 297)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 299) WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 300) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 301)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 302) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 303) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In yet another embodiment, the anti-PDL1 antibody is MPDL3280A (CASRegistry Number 1422185-06-5). In a still further embodiment, providedis an isolated anti-PDL1 antibody comprising a heavy chain variableregion comprising the heavy chain variable region amino acid sequencefrom SEQ ID NO:24 or SEQ ID NO:28 and/or a light chain variable regioncomprising the light chain variable region amino acid sequence from SEQID NO:21. In a still further embodiment, provided is an isolatedanti-PDL1 antibody comprising a heavy chain and/or a light chainsequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:

(SEQ ID NO: 278) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG,or(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 279) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In a still further embodiment, the invention provides for compositionscomprising any of the above described anti-PDL1 antibodies incombination with at least one pharmaceutically acceptable carrier.

In a still further embodiment, provided is an isolated nucleic acidencoding a light chain or a heavy chain variable region sequence of ananti-PDL1 antibody, wherein:

(a) the heavy chain further comprises and HVR-H1, HVR-H2 and an HVRH3sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ IDNO:289), AWISPYGGSTYYADSVKG (SEQ ID NO:290) and RHWPGGFDY (SEQ IDNO:291), respectively, and

(b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ IDNO:292), SASFLYS (SEQ ID NO:293) and QQYLYHPAT (SEQ ID NO:294),respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In aspect, theheavy chain variable region comprises one or more framework sequencesjuxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LCFR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework.

In a still further aspect, one or more of the heavy chain frameworksequences is the following:

HC-FR1 (SEQ ID NO: 295) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2(SEQ ID NO: 296) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 297)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 298) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 300) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 301)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 302) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 303) FGQGTKVEIKR.

In a still further specific aspect, the antibody described herein (suchas an anti-PD-1 antibody, an anti-PDL1 antibody, or an anti-PDL2antibody) further comprises a human or murine constant region. In astill further aspect, the human constant region is selected from thegroup consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still furtherspecific aspect, the human constant region is IgG1. In a still furtheraspect, the murine constant region is selected from the group consistingof IgG1, IgG2A, IgG2B, IgG3. In a still further aspect, the murineconstant region if IgG2A. In a still further specific aspect, theantibody has reduced or minimal effector function. In a still furtherspecific aspect, the minimal effector function results from productionin prokaryotic cells. In a still further specific aspect the minimaleffector function results from an “effector-less Fc mutation” oraglycosylation. In still a further aspect, the effector-less Fc mutationis an N297A or D265A/N297A substitution in the constant region.

In a still further aspect, provided herein are nucleic acids encodingany of the antibodies described herein. In some embodiments, the nucleicacid further comprises a vector suitable for expression of the nucleicacid encoding any of the previously described anti-PDL1, anti-PD-1, oranti-PDL2 antibodies. In a still further specific aspect, the vectorfurther comprises a host cell suitable for expression of the nucleicacid. In a still further specific aspect, the host cell is a eukaryoticcell or a prokaryotic cell. In a still further specific aspect, theeukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary(CHO).

The antibody or antigen binding fragment thereof, may be made usingmethods known in the art, for example, by a process comprising culturinga host cell containing nucleic acid encoding any of the previouslydescribed anti-PDL1, anti-PD-1, or anti-PDL2 antibodies orantigen-binding fragment in a form suitable for expression, underconditions suitable to produce such antibody or fragment, and recoveringthe antibody or fragment.

In some embodiments, the isolated anti-PDL1 antibody is aglycosylated.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Removal of glycosylation sites form anantibody is conveniently accomplished by altering the amino acidsequence such that one of the above-described tripeptide sequences (forN-linked glycosylation sites) is removed. The alteration may be made bysubstitution of an asparagine, serine or threonine residue within theglycosylation site another amino acid residue (e.g., glycine, alanine ora conservative substitution).

In any of the embodiments herein, the isolated anti-PDL1 antibody canbind to a human PDL1, for example a human PDL1 as shown inUniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof.

In a still further embodiment, the invention provides for a compositioncomprising an anti-PDL1, an anti-PD-1, or an anti-PDL2 antibody orantigen binding fragment thereof as provided herein and at least onepharmaceutically acceptable carrier. In some embodiments, the anti-PDL1,anti-PD-1, or anti-PDL2 antibody or antigen binding fragment thereofadministered to the individual is a composition comprising one or morepharmaceutically acceptable carrier.

Any of the pharmaceutically acceptable carriers described herein orknown in the art may be used.

In some embodiments, the anti-PDL1 antibody described herein is in aformulation comprising the antibody at an amount of about 60 mg/mL,histidine acetate in a concentration of about 20 mM, sucrose in aconcentration of about 120 mM, and polysorbate (e.g., polysorbate 20) ina concentration of 0.04% (w/v), and the formulation has a pH of about5.8. In some embodiments, the anti-PDL1 antibody described herein is ina formulation comprising the antibody in an amount of about 125 mg/mL,histidine acetate in a concentration of about 20 mM, sucrose is in aconcentration of about 240 mM, and polysorbate (e.g., polysorbate 20) ina concentration of 0.02% (w/v), and the formulation has a pH of about5.5.

Exemplary TIM3 Antagonists for Use in the Invention

Provided herein are methods for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a T cell activating bispecific antigen bindingmolecule, a PD-1 axis binding antagonist, and a TIM-3 antagonist. In oneembodiment, the TIM-3 antagonist is an anti-TIM-3 antibody. In someembodiments, the anti-TIM3 induces internalization of TIM3 expressed ona cell of at least 45% after 120 Minutes at 37° C. as determined by FACSanalysis. The cell is, e.g., a RPMI8226 cells (ATCC® CCL-155™). In oneembodiment, the antibody induces internalization of TIM3 on TIM3expressing RPMI8226 cells (ATCC® CCL-155™) of at least 55% after 120Minutes at 37° C. as determined by FACS analysis. In one embodiment, theantibody induces internalization of TIM3 on TIM3 expressing RPMI8226cells (ATCC® CCL-155™) of at least 60% after 240 Minutes at 37° C. asdetermined by FACS analysis. In one embodiment, the antibody inducesinternalization of TIM3 on TIM3 expressing RPMI8226 cells (ATCC®CCL-155™) of at least 65% after 240 Minutes at 37° C. as determined byFACS analysis.

In some embodiments, the anti-TIM3 antibody competes for binding to TIM3with an anti-Tim3 antibody comprising the VH and VL of Tim3_0016. Insome embodiments, the anti-TIM3 antibody binds to a human andcynomolgoues TIM3. In some embodiments, the anti-TIM3 antibody shows asa immunoconjugate a cytotoxic activity on TIM3 expressing cells. In onesuch embodiment, the immunoconjugate has a relative IC50 value of thecytotoxic activity as Pseudomonas exotoxin A conjugate on RPMI-8226cells of 0.1 or lower. In one embodiment, the anti-TIM3 antibody inducesinterferon-gamma release as determined by MLR assay.

In certain embodiments, the anti-TIM3 antibody binds to a human andcynomolgoues TIM3 and induces interferon-gamma release as determined bya MLR assay.

In one embodiment, the anti-TIM3 antibody comprises at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:304; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:305; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:306; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:307; or HVR-L1 comprising the amino acid sequence of SEQ IDNO:314; HVR-L1 comprising the amino acid sequence of SEQ ID NO:315; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:308; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:304; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:305; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:306; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:307; or HVR-L1comprising the amino acid sequence of SEQ ID NO:314; or HVR-L1comprising the amino acid sequence of SEQ ID NO:315; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:308; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:304; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:305; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:306; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:307; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:308; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:304; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:305; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:306; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:314; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:308; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:304; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:305; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:306; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:315; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:308; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:304, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:305,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:306; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:307; or HVR-L1 comprising the amino acidsequence of SEQ ID NO:314; or HVR-L1 comprising the amino acid sequenceof SEQ ID NO:315; (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:308 and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:304, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:305,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:306; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:307; or HVR-L1 comprising the amino acidsequence of SEQ ID NO:314; or HVR-L1 comprising the amino acid sequenceof SEQ ID NO:315; (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:308 and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:304, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:305,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:306; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:307; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:308 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:304, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:305,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:306; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:314; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:308 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:309.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:304, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:305,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:306; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:315; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:308 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:309.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:310 and a VL sequence of        SEQ ID NO:311;    -   ii) comprises a VH sequence of SEQ ID NO:312 and a VL sequence        of SEQ ID NO:313;    -   iii) or humanized variant of the VH and VL of the antibody        under i) or ii).

In one embodiment, the anti-TIM3 antibody comprises at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:316; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:317; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:318; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:319; (e) HVR-L2 comprising the amino acid sequence of SEQID NO:320; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:321.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:316; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:317; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:318; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:319; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:320; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:321.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:316, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:317,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:318; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:319; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:320 and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:321.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:316, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:317,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:318; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:319; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:320 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:321.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:322 and a VL sequence of        SEQ ID NO:323;    -   ii) or humanized variant of the VH and VL of the antibody under        i).

In one embodiment, the anti-TIM3 antibody comprises at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:324; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:325; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:326; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:327; (e) HVR-L2 comprising the amino acid sequence of SEQID NO:328; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:329.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:324; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:325; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:326; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:327; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:328; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:329.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:324, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:325,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:326; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:327; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:328 and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:329.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:324, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:325,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:326; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:327; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:328 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:329.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:330 and a VL sequence of        SEQ ID NO:331;    -   ii) or humanized variant of the VH and VL of the antibody under        i).

In one embodiment, the anti-TIM3 antibody comprises at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:332; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:333; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:334; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:335; (e) HVR-L2 comprising the amino acid sequence of SEQID NO:336; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:337.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:332; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:333; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:334; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:335; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:336; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:337.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:332, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:333,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:334; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:335; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:336 and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:337.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:332, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:333,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:334; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:335; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:336 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:337.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:338 and a VL sequence of        SEQ ID NO:339;    -   ii) or humanized variant of the VH and VL of the antibody under        i).

In one aspect, the invention provides an anti-TIM3 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:340; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:341; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:342; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:343; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:344; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:345.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:340; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:341; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:342; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:343; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:344; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:345.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:340, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:341,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:342; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:343; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:344 and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:345.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:340, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:341,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:342; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:343; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:344 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:345.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:346 and a VL sequence of        SEQ ID NO:347;    -   ii) or humanized variant of the VH and VL of the antibody under        i).

In one embodiment, the anti-TIM3 antibody comprises at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:348; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:349; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:350; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:351; (e) HVR-L2 comprising the amino acid sequence of SEQID NO:352; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:353.

In one aspect, the invention provides an anti-TIM3 antibody comprising(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:348; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:349; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:350; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:351; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:352; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:353.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:348, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:349,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:350; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:351; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:352 and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:353.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:348, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:349,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:350; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:351; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:352 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:353.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:354 and a VL sequence of        SEQ ID NO:355;    -   ii) or humanized variant of the VH and VL of the antibody under        i).

In one embodiment, the anti-TIM3 antibody comprises at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:356; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:357; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:358; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:359; (e) HVR-L2 comprising the amino acid sequence of SEQID NO:360; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:361.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:356; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:357; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:358; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:359; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:360; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:361.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:356, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:357, and (iii) HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO: 358; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 359; (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:360 and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO:361.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:356, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 357,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO: 358; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 359; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:360 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:361.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:362 and a VL sequence of        SEQ ID NO:363;    -   ii) or humanized variant of the VH and VL of the antibody under        i).

In one embodiment, the anti-TIM3 antibody comprises at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:364; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:365; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:366; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:367; (e) HVR-L2 comprising the amino acid sequence of SEQID NO:368; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:369.

In one embodiment, the anti-TIM3 antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:364; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:365; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:366; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:367; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:368; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:369.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising at least one, at least two, or all three VH HVR sequencesselected from (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:364, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:365,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:366; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:367; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:368 and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:369.

In one embodiment, the anti-TIM3 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:364, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:365,and (iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:366; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:367; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:368 and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:369.

In one embodiment such anti-TIM3 antibody comprises

-   -   i) comprises a VH sequence of SEQ ID NO:370 and a VL sequence of        SEQ ID NO:371;    -   ii) or humanized variant of the VH and VL of the antibody under        i).

In any of the above embodiments, an anti-TIM3 antibody is humanized. Inone embodiment, an anti-TIM3 antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework. Inanother embodiment, an anti-TIM3 antibody comprises HVRs as in any ofthe above embodiments, and further comprises a VH and VL comprising suchHVRs. In a further aspect, the anti-TIM3 antibody binds to the sameepitope as an anti-TIM3 antibody provided herein. For example, incertain embodiments, anti-TIM3 antibody binds to the same epitope asanti-TIM3 antibody comprising a VH sequence of SEQ ID NO:310 and a VLsequence of SEQ ID NO:311, or anti-TIM3 antibody binds to the sameepitope as anti-TIM3 antibody comprising a VH sequence of SEQ ID NO:312and a VL sequence of SEQ ID NO:313, or an antibody is provided thatbinds to the same epitope as anti-TIM3 antibody comprising a VH sequenceof SEQ ID NO:322 and a VL sequence of SEQ ID NO:323, or an antibody isprovided that binds to the same epitope as anti-TIM3 antibody comprisinga VH sequence of SEQ ID NO:330 and a VL sequence of SEQ ID NO:331, or anantibody is provided that binds to the same epitope as anti-TIM3antibody comprising a VH sequence of SEQ ID NO:338 and a VL sequence ofSEQ ID N0339, or an antibody is provided that binds to the same epitopeas anti-TIM3 antibody comprising a VH sequence of SEQ ID NO:346 and a VLsequence of SEQ ID NO:347, or an antibody is provided that binds to thesame epitope as anti-TIM3 antibody comprising a VH sequence of SEQ IDNO:354 and a VL sequence of SEQ ID NO:355, or an antibody is providedthat binds to the same epitope as anti-TIM3 antibody comprising a VHsequence of SEQ ID NO:362 and a VL sequence of SEQ ID NO:363, or anantibody is provided that binds to the same epitope as anti-TIM3antibody comprising a VH sequence of SEQ ID NO:370 and a VL sequence ofSEQ ID NO:371. In one preferred embodiment an antibody is provided thatbinds to the same epitope as an anti-TIM3 antibody comprising a VHsequence of SEQ ID NO:310 and a VL sequence of SEQ ID NO:311.

In one embodiment, the anti-TIM3 competes for binding to human TIM3 withan anti-TIM3 antibody comprising a VH sequence of SEQ ID NO:310 and a VLsequence of SEQ ID NO:311 as determined in a competition assay usingTIM3 expressing RPMI-8226 cells (ATCC® CCL-155™).

In one embodiment, the anti-TIM3 antibody according to any of the aboveembodiments is a monoclonal antibody, including a chimeric, humanized orhuman antibody. In one embodiment, an anti-TIM3 antibody is an antibodyfragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂ fragment. Inanother embodiment, the antibody is a full length antibody, e.g., anintact IgG1 or IgG4 antibody or other antibody class or isotype asdefined herein.

In a further aspect, an anti-TIM3 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described herein.

In one embodiment, the anti-TIM3 antibody is any of the antibodiesdescribed in WO 2011/155607, WO 2013/006490, WO 03/063792, WO2009/097394, or WO 2011/159877. In one embodiment, the anti-TIM3antibody is F38-2E2. In some embodiments, the anti-TIM-3 antibodies areantibodies from hybridomas 8B.2C12 and 25F.1D6 and prepared as disclosedin U. S. Patent application Nos: 2004/0005322 and 2005/0191721, Sabatos,C. A. et al., Nature Immunol. 4:1102-1110, 2003, and Sanchez-Fueyo, A.et al., Nature Immunol. 4:1093-1012003, all of which are herebyincorporated by reference as if set forth in their entirety. Otherantibodies to TIM-3 are specifically contemplated and can be produced,e.g., with the methods disclosed herein. The nucleotide and proteinsequences of TIM3 human sequences can be found at Genbank accessionnumber AF251707.1 and Uniprot accession number Q8TDQ0. An exemplaryhuman TIM3 amino acid sequence is set forth at SEQ ID NO:380; anexemplary human TIM3 extracellular domain amino acid sequence is setforth at SEQ ID NO:381.

Antibody Preparation

As described above, in some embodiments, the PD-1 binding antagonist isan antibody (e.g., an anti-PD-1 antibody, an anti-PDL1 antibody, or ananti-PDL2 antibody). In some embodiments, the TIM3 antagonist is anantibody (e.g., an anti-TIM3 antagonist antibody). The antibodiesdescribed herein may be prepared using techniques available in the artfor generating antibodies, exemplary methods of which are described inmore detail in the following sections.

The antibody is directed against an antigen of interest. For example,the antibody may be directed against PD-1 (such as human PD-1), PDL1(such as human PDL1), PDL2 (such as human PDL2), an TIM3 (such as humanTIM3). Preferably, the antigen is a biologically important polypeptideand administration of the antibody to a mammal suffering from a disordercan result in a therapeutic benefit in that mammal.

In certain embodiments, an antibody described herein has a dissociationconstant (Kd) of 1 μM, 150 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01nM, or 0.001 nM (e.g. 10-8 M or less, e.g. from 10-8 M to 10-13 M, e.g.,from 10-9 M to 10-13 M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (125I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125I]-antigen aremixed with serial dilutions of a Fab of interest. The Fab of interest isthen incubated overnight; however, the incubation may continue for alonger period (e.g., about 65 hours) to ensure that equilibrium isreached. Thereafter, the mixtures are transferred to the capture platefor incubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT TM gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein.

Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μl/min. Association rates (kon) and dissociation rates(koff) are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen etal., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1s-1 by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

In some embodiments, an anti-TIM3 antibody as described herein exhibitsa binding affinity of at least 100 pM or less against human TIM3, abinding affinity of at least 300 pM or less against human TIM3, abinding affinity of at least 400 pM or less against human TIM3, aneutralizing ability of at least 40 nM or less against the human TIM3, aneutralizing ability of at least 120 nM or less against the human TIM3,and a neutralizing ability of at least 31 nM or less against the humanTIM3. In these embodiments, binding affinity may be measured by surfaceplasmon resonance as described in U.S. Pat. No. 8,771,697,

Antibody Fragments

In certain embodiments, an antibody described herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)2 fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat. Med 9:129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

Chimeric and Humanized Antibodies

In certain embodiments, an antibody described herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Nat. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof. In certain embodiments, a chimericantibody is a humanized antibody. Typically, a non-human antibody ishumanized to reduce immunogenicity to humans, while retaining thespecificity and affinity of the parental non-human antibody. Generally,a humanized antibody comprises one or more variable domains in whichHVRs, e.g., CDRs, (or portions thereof) are derived from a non-humanantibody, and FRs (or portions thereof) are derived from human antibodysequences. A humanized antibody optionally will also comprise at least aportion of a human constant region. In some embodiments, some FRresidues in a humanized antibody are substituted with correspondingresidues from a non-human antibody (e.g., the antibody from which theHVR residues are derived), e.g., to restore or improve antibodyspecificity or affinity. Humanized antibodies and methods of making themare reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633(2008), and are further described, e.g., in Riechmann et al., Nature332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321,and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Nat. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

Human Antibodies

In certain embodiments, an antibody described herein is a humanantibody. Human antibodies can be produced using various techniquesknown in the art. Human antibodies are described generally in van Dijkand van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg,Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region. Human antibodies can also be made byhybridoma-based methods. Human myeloma and mouse-human heteromyelomacell lines for the production of human monoclonal antibodies have beendescribed. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147: 86 (1991).) Human antibodies generated via human B-cellhybridoma technology are also described in Li et al., Proc. Nat. Acad.Sci. USA, 103:3557-3562 (2006). Additional methods include thosedescribed, for example, in U.S. Pat. No. 7,189,826 (describingproduction of monoclonal human IgM antibodies from hybridoma cell lines)and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing humanhybridomas). Human hybridoma technology (Trioma technology) is alsodescribed in Vollmers and Brandlein, Histology and Histopathology,20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings inExperimental and Clinical Pharmacology, 27(3):185-91 (2005). Humanantibodies may also be generated by isolating Fv clone variable domainsequences selected from human-derived phage display libraries. Suchvariable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Library-Derived Antibodies

Antibodies may be isolated by screening combinatorial libraries forantibodies with the desired activity or activities. For example, avariety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360. Antibodies or antibodyfragments isolated from human antibody libraries are considered humanantibodies or human antibody fragments herein.

Multispecific Antibodies

In certain embodiments, an antibody described herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. Examples of T cell activating bispecific antigenbinding molecules specific for FolR1 and CD3 are described herein. Insome embodiments, the PD1 axis component antagonist is multispecific. Inone of the binding specificities is for a PD-1 axis component (e.g.,PD-1, PDL1, or PDL2) and the other is for any other antigen. In someembodiments, one of the binding specificities is for IL-17 or IL-17R andthe other is for any other antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of a PD-1 axis component(e.g., PD-1, PDL1, or PDL2), IL-17, or IL-17R. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

In some embodiments, one of the binding specificities is for a PD-1 axiscomponent (e.g., PD-1, PDL1, or PDL2) and the other is for IL-17 orIL-17R. Provided herein are methods for treating or delaying progressionof cancer in an individual comprising administering to the individual aneffective amount of a multispecific antibody, wherein the multispecificantibody comprises a first binding specificity for a PD-1 axis component(e.g., PD-1, PDL1, or PDL2) and a second binding specificity for IL-17or IL-17R. In some embodiments, a multispecific antibody may be made byany of the techniques described herein and below.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); crosslinking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:64446448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1). The antibody or fragment herein also includes a“Dual Acting FAb” or “DAF” comprising an antigen binding site that bindsto a PD-1 axis component (e.g., PD-1, PDL1, or PDL2), IL-17, or IL-17Ras well as another, different antigen (see, US 2008/0069820, forexample).

C. Nucleic Acid Sequences, Vectors and Methods of Production

Polynucleotides encoding a T cell activating bispecific antigen bindingmolecule, e.g., a T cell activating bispecific antigen binding moleculecomprising a first antigen binding site specific for Folate Receptor 1(FolR1) and a second antigen binding site specific for CD3, andantibodies may be used for production of the T cell activatingbispecific antigen binding molecule and antibodies described herein. TheT cell activating bispecific antigen binding molecule and antibodies ofthe invention may be expressed as a single polynucleotide that encodesthe entire bispecific antigen binding molecule or as multiple (e.g., twoor more) polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional T cell activatingbispecific antigen binding molecule and antibody. For example, the lightchain portion of a Fab fragment may be encoded by a separatepolynucleotide from the portion of the bispecific antibody or theantibody binding to FolR1 comprising the heavy chain portion of the Fabfragment, an Fc domain subunit and optionally (part of) another Fabfragment. When co-expressed, the heavy chain polypeptides will associatewith the light chain polypeptides to form the Fab fragment. In anotherexample, the portion of the T cell activating bispecific antigen bindingmolecule or the FolR1 antigen binding portion provided thereincomprising one of the two Fc domain subunits and optionally (part of)one or more Fab fragments could be encoded by a separate polynucleotidefrom the portion of the bispecific antibody or the antibody binding toFolR1 provided therein comprising the other of the two Fc domainsubunits and optionally (part of) a Fab fragment. When co-expressed, theFc domain subunits will associate to form the Fc domain.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

D. Antibody Variants

In certain embodiments, amino acid sequence variants of the T cellactivating bispecific antigen binding molecule specific for FolR1 andCD3 provided herein and antibodies are contemplated, in addition tothose described above. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the T cellactivating bispecific antigen binding molecule. Amino acid sequencevariants of a T cell activating bispecific antigen binding molecule andantibody may be prepared by introducing appropriate modifications intothe nucleotide sequence encoding the T cell activating bispecificantigen binding molecule or antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of residues within the amino acid sequences ofthe antibody. Any combination of deletion, insertion, and substitutioncan be made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

1. Substitution, Insertion, and Deletion Variants

In certain embodiments, variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table B under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table B under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE B Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gn;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

2. Glycosylation Variants

In certain embodiments, a T cell activating bispecific antigen bindingmolecule or an antibody provided herein is altered to increase ordecrease the extent to which the antibody is glycosylated. Addition ordeletion of glycosylation sites to an antibody may be convenientlyaccomplished by altering the amino acid sequence such that one or moreglycosylation sites is created or removed.

Where the T cell activating bispecific antigen binding molecule or theantibody used with the invention comprises an Fc region, thecarbohydrate attached thereto may be altered. Native antibodies producedby mammalian cells typically comprise a branched, biantennaryoligosaccharide that is generally attached by an N-linkage to Asn297 ofthe CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH15:26-32 (1997). The oligosaccharide may include various carbohydrates,e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialicacid, as well as a fucose attached to a GlcNAc in the “stem” of thebiantennary oligosaccharide structure. In some embodiments,modifications of the oligosaccharide in a bispecific antibody or anantibody binding to DR5 of the invention may be made in order to createantibody variants with certain improved properties.

In one embodiment, bispecific antibody variants or variants ofantibodies are provided having a carbohydrate structure that lacksfucose attached (directly or indirectly) to an Fc region. For example,the amount of fucose in such antibody may be from 1% to 80%, from 1% to65%, from 5% to 65% or from 20% to 40%. The amount of fucose isdetermined by calculating the average amount of fucose within the sugarchain at Asn297, relative to the sum of all glycostructures attached toAsn 297 (e. g. complex, hybrid and high mannose structures) as measuredby MALDI-TOF mass spectrometry, as described in WO 2008/077546, forexample. Asn297 refers to the asparagine residue located at aboutposition 297 in the Fc region (Eu numbering of Fc region residues);however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants mayhave improved ADCC function. See, e.g., US Patent Publication Nos. US2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl NoUS 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

T cell activating bispecific antigen binding molecule variants andantibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the T cell activating bispecific antigen binding molecule binding toFolR1 is bisected by GlcNAc. Such T cell activating bispecific antigenbinding molecule variants may have reduced fucosylation and/or improvedADCC function. Examples of such antibody variants are described, e.g.,in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umanaet al.); and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

3. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered T cell activating bispecific antigen binding molecule andantibodies, e.g., THIOMABS, in which one or more residues of the T cellactivating bispecific antigen binding molecule are substituted withcysteine residues. In particular embodiments, the substituted residuesoccur at accessible sites of the T cell activating bispecific antigenbinding molecule. By substituting those residues with cysteine, reactivethiol groups are thereby positioned at accessible sites of the antibodyand may be used to conjugate the antibody to other moieties, such asdrug moieties or linker-drug moieties, to create an immunoconjugate. Incertain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region. Cysteine engineered antibodies may be generatedas described, e.g., in U.S. Pat. No. 7,521,541.

E. Recombinant Methods and Compositions

T cell activating bispecific antigen binding molecule and antibodies ofthe invention may be obtained, for example, by solid-state peptidesynthesis (e.g. Merrifield solid phase synthesis) or recombinantproduction. For recombinant production one or more polynucleotideencoding the T cell activating bispecific antigen binding molecule orantibodies (or fragments), e.g., as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such polynucleotide may be readily isolated andsequenced using conventional procedures. In one embodiment a vector,preferably an expression vector, comprising one or more of thepolynucleotides of the invention is provided. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing the coding sequence of a T cell activating bispecificantigen binding molecule or an antibody along with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WileyInterscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encoding Tcell activating bispecific antigen binding molecule (fragment) or anantibody (fragment) (i.e. the coding region) is cloned in operableassociation with a promoter and/or other transcription or translationcontrol elements. As used herein, a “coding region” is a portion ofnucleic acid which consists of codons translated into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is not translated into anamino acid, it may be considered to be part of a coding region, ifpresent, but any flanking sequences, for example promoters, ribosomebinding sites, transcriptional terminators, introns, 5′ and 3′untranslated regions, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g. on a single vector, or in separate polynucleotideconstructs, e.g. on separate (different) vectors. Furthermore, anyvector may contain a single coding region, or may comprise two or morecoding regions, e.g. a vector of the present invention may encode one ormore polypeptides, which are post- or co-translationally separated intothe final proteins via proteolytic cleavage. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a polynucleotide encoding theT cell activating bispecific antigen binding molecule (fragment) or anantibody, or variant or derivative thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells.

Other transcription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit â-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the T cell activating bispecific antigen binding molecule or theantibody is desired, DNA encoding a signal sequence may be placedupstream of the nucleic acid encoding a bispecific antibody of theinvention or the antibody binding to DR5 of the invention or a fragmentthereof. According to the signal hypothesis, proteins secreted bymammalian cells have a signal peptide or secretory leader sequence whichis cleaved from the mature protein once export of the growing proteinchain across the rough endoplasmic reticulum has been initiated. Thoseof ordinary skill in the art are aware that polypeptides secreted byvertebrate cells generally have a signal peptide fused to the N-terminusof the polypeptide, which is cleaved from the translated polypeptide toproduce a secreted or “mature” form of the polypeptide. In certainembodiments, the native signal peptide, e.g. an immunoglobulin heavychain or light chain signal peptide is used, or a functional derivativeof that sequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling the Tcell activating bispecific antigen binding molecule may be includedwithin or at the ends of the T cell activating bispecific antigenbinding molecule (fragment) or the antibody (fragment) encodingpolynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes a T cell activating bispecificantigen binding molecule or an antibody of the invention or a partthereof. As used herein, the term “host cell” refers to any kind ofcellular system which can be engineered to generate the T cellactivating bispecific antigen binding molecule, e.g., the FolR1 T cellactivating bispecific antigen binding molecules disclosed herein, orantibody, e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, andanti-TIM3 antibodies of the invention or fragments thereof. Host cellssuitable for replicating and for supporting expression of T cellactivating bispecific antigen binding molecule and antibodies of theinvention are well known in the art. Such cells may be transfected ortransduced as appropriate with the particular expression vector andlarge quantities of vector containing cells can be grown for seedinglarge scale fermenters to obtain sufficient quantities of the T cellactivating bispecific antigen binding molecule and antibodies forclinical applications. Suitable host cells include prokaryoticmicroorganisms, such as E. coli, or various eukaryotic cells, such asChinese hamster ovary cells (CHO), insect cells, or the like. Forexample, polypeptides may be produced in bacteria in particular whenglycosylation is not needed. After expression, the polypeptide may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified. In addition to prokaryotes, eukaryotic microbes suchas filamentous fungi or yeast are suitable cloning or expression hostsfor polypeptide-encoding vectors, including fungi and yeast strainswhose glycosylation pathways have been “humanized”, resulting in theproduction of a polypeptide with a partially or fully humanglycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004),and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells forthe expression of (glycosylated) polypeptides are also derived frommulticellular organisms (invertebrates and vertebrates). Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains have been identified which may be used in conjunction withinsect cells, particularly for transfection of Spodoptera frugiperdacells. Plant cell cultures can also be utilized as hosts. See e.g. U.S.Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants). Vertebrate cells may also be used as hosts. Forexample, mammalian cell lines that are adapted to grow in suspension maybe useful. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line(293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)),monkey kidney cells (CV1), African green monkey kidney cells (VERO-76),human cervical carcinoma cells (HELA), canine kidney cells (MDCK),buffalo rat liver cells (BRL 3A), human lung cells (W138), human livercells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (asdescribed, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68(1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host celllines include Chinese hamster ovary (CHO) cells, including dhfr CHOcells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); andmyeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review ofcertain mammalian host cell lines suitable for protein production, see,e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo,ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cells includecultured cells, e.g., mammalian cultured cells, yeast cells, insectcells, bacterial cells and plant cells, to name only a few, but alsocells comprised within a transgenic animal, transgenic plant or culturedplant or animal tissue. In one embodiment, the host cell is a eukaryoticcell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO)cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., YO,NS0, Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be used in the T cell activatingbispecific antigen binding molecules of the invention. Non-limitingantibodies, antibody fragments, antigen binding domains or variableregions useful in the present invention can be of murine, primate, orhuman origin. If the T cell activating bispecific antigen bindingmolecule is intended for human use, a chimeric form of antibody may beused wherein the constant regions of the antibody are from a human. Ahumanized or fully human form of the antibody can also be prepared inaccordance with methods well known in the art (see e. g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988);Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Humanantibodies and human variable regions can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) andLonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regionscan form part of and be derived from human monoclonal antibodies made bythe hybridoma method (see e.g. Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Human antibodies and human variable regions may also be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge (see e.g. Lonberg,Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variableregions may also be generated by isolating Fv clone variable regionsequences selected from human-derived phage display libraries (see e.g.,Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al.,Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv (scFv)fragments or as Fab fragments.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in U.S. Pat. Appl.Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the T cell activatingbispecific antigen binding molecule of the invention to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). Competition assays may be used to identify an antibody,antibody fragment, antigen binding domain or variable domain thatcompetes with a reference antibody for binding to a particular antigen,e.g. an antibody that competes with the V9 antibody for binding to CD3.In certain embodiments, such a competing antibody binds to the sameepitope (e.g. a linear or a conformational epitope) that is bound by thereference antibody. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In an exemplary competition assay, immobilized antigen(e.g. CD3) is incubated in a solution comprising a first labeledantibody that binds to the antigen (e.g. V9 antibody, described in U.S.Pat. No. 6,054,297) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to theantigen. The second antibody may be present in a hybridoma supernatant.As a control, immobilized antigen is incubated in a solution comprisingthe first labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto the antigen, excess unbound antibody is removed, and the amount oflabel associated with immobilized antigen is measured. If the amount oflabel associated with immobilized antigen is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding tothe antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manualch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in U.S. Pat. Appl.Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the T cell activatingbispecific antigen binding molecule or the antibody of the invention tobind to a specific antigenic determinant can be measured either throughan enzyme-linked immunosorbent assay (ELISA) or other techniquesfamiliar to one of skill in the art, e.g. surface plasmon resonancetechnique (analyzed on a BIACORE T100 system) (Liljeblad, et al., GlycoJ 17, 323-329 (2000)), and traditional binding assays (Heeley, EndocrRes 28, 217-229 (2002)). Competition assays may be used to identify anantibody, antibody fragment, antigen binding domain or variable domainthat competes with a reference antibody for binding to a particularantigen. In certain embodiments, such a competing antibody binds to thesame epitope (e.g. a linear or a conformational epitope) that is boundby the reference antibody. Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.). In an exemplary competition assay,immobilized antigen is incubated in a solution comprising a firstlabeled antibody that binds to the antigen and a second unlabeledantibody that is being tested for its ability to compete with the firstantibody for binding to the antigen. The second antibody may be presentin a hybridoma supernatant. As a control, immobilized antigen isincubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody.

After incubation under conditions permissive for binding of the firstantibody to the antigen, excess unbound antibody is removed, and theamount of label associated with immobilized antigen is measured. If theamount of label associated with immobilized antigen is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to the antigen. See Harlow and Lane (1988) Antibodies: ALaboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

T cell activating bispecific antigen binding molecules and antibodiesprepared as described herein may be purified by art-known techniquessuch as high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, affinity chromatography, sizeexclusion chromatography, and the like. The actual conditions used topurify a particular protein will depend, in part, on factors such as netcharge, hydrophobicity, hydrophilicity etc., and will be apparent tothose having skill in the art. For affinity chromatography purificationan antibody, ligand, receptor or antigen can be used to which thebispecific antibody or the antibody binding to DR5 binds. For example,for affinity chromatography purification of bispecific antibodies of theinvention, a matrix with protein A or protein G may be used. SequentialProtein A or G affinity chromatography and size exclusion chromatographycan be used to isolate a bispecific antibody essentially as described inthe Examples. The purity of the bispecific antibody or the antibodybinding to DR5 can be determined by any of a variety of well-knownanalytical methods including gel electrophoresis, high pressure liquidchromatography, and the like.

F. Assays

T cell activating bispecific antigen binding molecules, e.g., a T cellactivating bispecific antigen binding molecules comprising a firstantigen binding site specific for Folate Receptor 1 (FolR1) and a secondantigen binding site specific for CD3, and antibodies, e.g., anti-PD-1axis binding antagonist antibodies and anti-TIM3 antagonist antibodiesprovided herein may be identified, screened for, or characterized fortheir physical/chemical properties and/or biological activities byvarious assays known in the art.

1. Affinity Assays

The affinity of the T cell activating bispecific antigen bindingmolecules, e.g., a T cell activating bispecific antigen bindingmolecules comprising a first antigen binding site specific for FolateReceptor 1 (FolR1) and a second antigen binding site specific for CD3,and antibodies, e.g., anti-PD-1 axis binding antagonist antibodies andanti-TIM3 antagonist antibodies provided herein for their respectiveantigen, e.g., FolR1, PD-1, PD-L1, TIM3, can be determined in accordancewith the methods set forth in the Examples by surface plasmon resonance(SPR), using standard instrumentation such as a BIAcore instrument (GEHealthcare), and receptors or target proteins such as may be obtained byrecombinant expression. Alternatively, binding of T cell activatingbispecific antigen binding molecules and antibodies provided therein totheir respective antigen may be evaluated using cell lines expressingthe particular receptor or target antigen, for example by flow cytometry(FACS).

K_(D) may be measured by surface plasmon resonance using a BIACORE® T100machine (GE Healthcare) at 25° C. To analyze the interaction between theFc-portion and Fc receptors, His-tagged recombinant Fc-receptor iscaptured by an anti-Penta His antibody (Qiagen) (“Penta His” disclosedas SEQ ID NO: 392) immobilized on CM5 chips and the bispecificconstructs are used as analytes. Briefly, carboxymethylated dextranbiosensor chips (CM5, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti Penta-His antibody (“Penta His” disclosed as SEQ ID NO: 392) isdiluted with 10 mM sodium acetate, pH 5.0, to 40 μg/ml before injectionat a flow rate of 5 μl/min to achieve approximately 6500 response units(RU) of coupled protein. Following the injection of the ligand, 1 Methanolamine is injected to block unreacted groups. Subsequently theFc-receptor is captured for 60 s at 4 or 10 nM. For kineticmeasurements, four-fold serial dilutions of the bispecific construct(range between 500 nM and 4000 nM) are injected in HBS-EP (GEHealthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20,pH 7.4) at 25° C. at a flow rate of 30 μl/min for 120 s.

To determine the affinity to the target antigen, bispecific constructsare captured by an anti human Fab specific antibody (GE Healthcare) thatis immobilized on an activated CM5-sensor chip surface as described forthe anti Penta-His antibody (“Penta His” disclosed as SEQ ID NO: 392).The final amount of coupled protein is approximately 12000 RU. Thebispecific constructs are captured for 90 s at 300 nM. The targetantigens are passed through the flow cells for 180 s at a concentrationrange from 250 to 1000 nM with a flowrate of 30 μl/min. The dissociationis monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting theresponse obtained on reference flow cell. The steady state response wasused to derive the dissociation constant K_(D) by non-linear curvefitting of the Langmuir binding isotherm. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

2. Binding Assays and Other Assays

In one aspect, a T cell activating bispecific antigen binding molecules,e.g., a T cell activating bispecific antigen binding moleculescomprising a first antigen binding site specific for Folate Receptor 1(FolR1) and a second antigen binding site specific for CD3, andantibodies, e.g., anti-PD-1 axis binding antagonist antibodies andanti-TIM3 antagonist antibodies of the invention is tested for itsantigen binding activity, e.g., by known methods such as ELISA, Westernblot, etc.

In another aspect, competition assays may be used to identify anantibody or fragment that competes with a specific reference antibodyfor binding to the respective antigens. In certain embodiments, such acompeting antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by a specific reference antibody.Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris (1996) “Epitope Mapping Protocols,” inMethods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).Further methods are described in the example section.

3. Activity Assays

In one aspect, assays are provided for identifying T cell activatingbispecific antigen binding molecules, e.g., a T cell activatingbispecific antigen binding molecules comprising a first antigen bindingsite specific for Folate Receptor 1 (FolR1) and a second antigen bindingsite specific for CD3, and antibodies, e.g., anti-PD-1 axis bindingantagonist antibodies and anti-TIM3 antagonist antibodies providedherein having biological activity. Biological activity may include,e.g., inducing DNA fragmentation, induction of apoptosis and lysis oftargeted cells. Antibodies having such biological activity in vivoand/or in vitro are also provided.

In certain embodiments, T cell activating antigen binding molecule andantibody of the invention is tested for such biological activity. Assaysfor detecting cell lysis (e.g. by measurement of LDH release) orapoptosis (e.g. using the TUNEL assay) are well known in the art. Assaysfor measuring ADCC or CDC are also described in WO 2004/065540 (seeExample 1 therein), the entire content of which is incorporated hereinby reference.

G. Pharmaceutical Formulations

Pharmaceutical formulations of a T cell activating bispecific antigenbinding molecules, e.g., a T cell activating bispecific antigen bindingmolecule comprising a first antigen binding site specific for FolateReceptor 1 (FolR1) and a second antigen binding site specific for CD3,and antibodies, e.g., anti-PD-1 axis binding antagonist antibodies andanti-TIM3 antagonist antibodies as described herein are prepared bymixing such T cell activating bispecific antigen binding molecules orantibody having the desired degree of purity with one or more optionalpharmaceutically acceptable carriers (Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

H. Therapeutic Methods and Compositions

The therapeutic combinations comprising one or more of the T cellactivating bispecific antigen binding molecules and the anti-PD-1 axisbinding antagonist antibody and, optionally, the TIM3 antagonistprovided herein may be used in therapeutic methods.

In one aspect, a T cell activating bispecific antigen binding moleculesthat binds to Folate Receptor 1 (FolR1) and CD3 for use as a medicamentis provided for use in combination with an anti-PD-1 axis bindingantagonist antibody. In certain embodiments, a T cell activatingbispecific antigen binding molecules that binds to FolR1 and CD3 for usein combination with an anti-PD-1 axis binding antagonist antibody isprovided for use in a method of treatment. In certain embodiments, thecombination further comprises a TIM3 antagonist, e.g., an anti-TIM3antagonist antibody. In certain embodiments, the invention provides a Tcell activating bispecific antigen binding molecules that binds to FolR1and CD3 and an anti-PD-1 axis binding antagonist antibody for use in amethod of treating an individual having cancer comprising administeringto the individual an effective amount of the T cell activatingbispecific antigen binding molecules that binds to FolR1 and CD3 and theanti-PD-1 axis binding antagonist antibody. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one TIM3 antagonist, e.g., as described below. An“individual” according to any of the above embodiments is preferably ahuman. In one preferred embodiment, said cancer is pancreatic cancer,sarcoma or colorectal carcinoma. In other embodiments, the cancer iscolorectal cancer, sarcoma, head and neck cancers, squamous cellcarcinomas, breast cancer, pancreatic cancer, gastric cancer,non-small-cell lung carcinoma, small-cell lung cancer or mesothelioma.In embodiments in which the cancer is breast cancer, the breast cancermay be triple negative breast cancer.

In a further aspect, the invention provides the use of a therapeuticcombination comprising a T cell activating bispecific antigen bindingmolecules that binds to FolR1 and CD3 and an anti-PD-1 axis bindingantagonist antibody in the manufacture or preparation of a medicament.In one embodiment, the combination further comprises a TIM3 antagonist.In one embodiment, the medicament is for treatment of cancer. In afurther embodiment, the medicament is for use in a method of treatingcancer comprising administering to an individual having cancer aneffective amount of the medicament. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, e.g., as described below. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treatingcancer. In one embodiment, the method comprises administering to anindividual having cancer an effective amount of a therapeuticcombination comprising a T cell activating bispecific antigen bindingmolecules that binds to FolR1 and CD3 and an anti-PD-1 axis bindingantagonist antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, as described below. In one suchembodiment, the at least one additional therapeutic agent is ananti-TIM3 antagonist antibody. An “individual” according to any of theabove embodiments may be a human. In one preferred embodiment saidcancer is pancreatic cancer, sarcoma or colorectal carcinoma. In otherembodiments, the cancer is colorectal cancer, sarcoma, head and neckcancers, squamous cell carcinomas, breast cancer, pancreatic cancer,gastric cancer, non-small-cell lung carcinoma, small-cell lung cancer ormesothelioma.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the T cell activating bispecific antigen bindingmolecules that binds to FolR1 and CD3 provided herein, e.g., for use inany of the above therapeutic methods, and an anti-PD-1 axis bindingantagonist antibody. In one embodiment, a pharmaceutical formulationcomprises any of the T cell activating bispecific antigen bindingmolecules that binds to FolR1 provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of T cell activating bispecific antigen binding moleculesthat binds to FolR1 and CD3 and an anti-PD-1 axis binding antagonistantibody provided herein and at least one additional therapeutic agent,e.g., as described below.

A bispecific antibody can be administered by any suitable means,including parenteral, intrapulmonary, and intranasal, and, if desiredfor local treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Bispecific antibodies may be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thebispecific antibody need not be, but is optionally formulated with oneor more agents currently used to prevent or treat the disorder inquestion. The effective amount of such other agents depends on theamount of antibody present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage of abispecific antibody will depend on the type of disease to be treated,the type of antibody, the severity and course of the disease, whetherthe bispecific antibody is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the bispecific antibody and the discretion of the attendingphysician. The bispecific antibody is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1mg/kg-10 mg/kg) of the bispecific antibody or the novel antibody bindingto DR5 can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the bispecific would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg may be administered to the patient. Such doses maybe administered intermittently, e.g. every week or every three weeks(e.g. such that the patient receives from about two to about twenty, ore.g. about six doses of the bispecific antibody). An initial higherloading dose, followed by one or more lower doses may be administered.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to the T cell activating bispecific antigenbinding molecules that binds to FolR1 and CD3 and the anti-PD-1 axisbinding antagonist antibody, and, optionally, the anti-TIM3 antagonistantibody.

I. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a bispecific antibody and an additional active agent isthe further chemotherapeutic agent as described herein. The label orpackage insert indicates that the composition is used for treating thecondition of choice. Moreover, the article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises a bispecific antibody; and (b) a second containerwith a composition contained therein, wherein the composition comprisesa further cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto the T cell activating bispecific antigen binding molecules that bindsto FolR1 and CD3 and the anti-PD-1 axis binding antagonist antibody and,optionally, the anti-TIM3 antagonist antibody.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

General Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturers'instructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, 5^(th)ed., NIH Publication No. 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments where required were either generated by PCR usingappropriate templates or were synthesized by Geneart AG (Regensburg,Germany) from synthetic oligonucleotides and PCR products by automatedgene synthesis. In cases where no exact gene sequence was available,oligonucleotide primers were designed based on sequences from closesthomologues and the genes were isolated by RT-PCR from RNA originatingfrom the appropriate tissue. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow sub-cloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells.

Isolation of Primary Human Pan T Cells from PBMCs

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation from enriched lymphocyte preparations (buffycoats) obtained from local blood banks or from fresh blood from healthyhuman donors. Briefly, blood was diluted with sterile PBS and carefullylayered over a Histopaque gradient (Sigma, H8889). After centrifugationfor 30 minutes at 450×g at room temperature (brake switched off), partof the plasma above the PBMC containing interphase was discarded. ThePBMCs were transferred into new 50 ml Falcon tubes and tubes were filledup with PBS to a total volume of 50 ml. The mixture was centrifuged atroom temperature for 10 minutes at 400×g (brake switched on). Thesupernatant was discarded and the PBMC pellet washed twice with sterilePBS (centrifugation steps at 4° C. for 10 minutes at 350×g). Theresulting PBMC population was counted automatically (ViCell) and storedin RPMI640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine(Biochrom, K0302) at 37° C., 5% CO₂ in the incubator until assay start.

T cell enrichment from PBMCs was performed using the Pan T CellIsolation Kit II (Miltenyi Biotec #130-091-156), according to themanufacturer's instructions. Briefly, the cell pellets were diluted in40 μl cold buffer per 10 million cells (PBS with 0.5% BSA, 2 mM EDTA,sterile filtered) and incubated with 10 μl Biotin-Antibody Cocktail per10 million cells for 10 min at 4° C. 30 μl cold buffer and 20 μlAnti-Biotin magnetic beads per 10 million cells were added, and themixture incubated for another 15 min at 4° C. Cells were washed byadding 10-20× the current volume and a subsequent centrifugation step at300×g for 10 min. Up to 100 million cells were resuspended in 500 μlbuffer. Magnetic separation of unlabeled human pan T cells was performedusing LS columns (Miltenyi Biotec #130-042-401) according to themanufacturer's instructions. The resulting T cell population was countedautomatically (ViCell) and stored in AIM-V medium at 37° C., 5% CO₂ inthe incubator until assay start (not longer than 24 h).

Isolation of Primary Human Naive T Cells from PBMCs

Peripheral blood mononuclar cells (PBMCs) were prepared by Histopaquedensity centrifugation from enriched lymphocyte preparations (buffycoats) obtained from local blood banks or from fresh blood from healthyhuman donors. T-cell enrichment from PBMCs was performed using the NaiveCD8⁺ T cell isolation Kit from Miltenyi Biotec (#130-093-244), accordingto the manufacturer's instructions, but skipping the last isolation stepof CD8⁺ T cells (also see description for the isolation of primary humanpan T cells).

Isolation of Murine Pan T Cells from Splenocytes

Spleens were isolated from C57BL/6 mice, transferred into a GentleMACSC-tube (Miltenyi Biotech #130-093-237) containing MACS buffer (PBS+0.5%BSA+2 mM EDTA) and dissociated with the GentleMACS Dissociator to obtainsingle-cell suspensions according to the manufacturer's instructions.The cell suspension was passed through a pre-separation filter to removeremaining undissociated tissue particles. After centrifugation at 400×gfor 4 min at 4° C., ACK Lysis Buffer was added to lyse red blood cells(incubation for 5 min at room temperature). The remaining cells werewashed with MACS buffer twice, counted and used for the isolation ofmurine pan T cells. The negative (magnetic) selection was performedusing the Pan T Cell Isolation Kit from Miltenyi Biotec (#130-090-861),following the manufacturer's instructions. The resulting T cellpopulation was automatically counted (ViCell) and immediately used forfurther assays.

Isolation of Primary Cynomolgus PBMCs from Heparinized Blood

Peripheral blood mononuclar cells (PBMCs) were prepared by densitycentrifugation from fresh blood from healthy cynomolgus donors, asfollows: Heparinized blood was diluted 1:3 with sterile PBS, andLymphoprep medium (Axon Lab #1114545) was diluted to 90% with sterilePBS. Two volumes of the diluted blood were layered over one volume ofthe diluted density gradient and the PBMC fraction was separated bycentrifugation for 30 min at 520×g, without brake, at room temperature.The PBMC band was transferred into a fresh 50 ml Falcon tube and washedwith sterile PBS by centrifugation for 10 min at 400×g at 4° C. Onelow-speed centrifugation was performed to remove the platelets (15 minat 150×g, 4° C.), and the resulting PBMC population was automaticallycounted (ViCell) and immediately used for further assays.

Example 1 Purification of Biotinylated Folate Receptor-Fc Fusions

To generate new antibodies against human FolR1 the following antigensand screening tools were generated as monovalent Fc fusion proteins (theextracellular domain of the antigen linked to the hinge region ofFc-knob which is co-expressed with an Fc-hole molecule). The antigengenes were synthesized (Geneart, Regensburg, Germany) based on sequencesobtained from GenBank or SwissProt and inserted into expression vectorsto generate fusion proteins with Fc-knob with a C-terminal Avi-tag forin vivo or in vitro biotinylation. In vivo biotinylation was achieved byco-expression of the bacterial birA gene encoding a bacterial biotinligase during production. Expression of all genes was under control of achimeric MPSV promoter on a plasmid containing an oriP element forstable maintenance of the plasmids in EBNA containing cell lines.

For preparation of the biotinylated monomeric antigen/Fc fusionmolecules, exponentially growing suspension HEK293 EBNA cells wereco-transfected with three vectors encoding the two components of fusionprotein (knob and hole chains) as well as BirA, an enzyme necessary forthe biotinylation reaction. The corresponding vectors were used at a9.5:9.5:1 ratio (“antigen ECD-Fc knob-avi tag”: “Fc hole”: “BirA”).

For protein production in 500 ml shake flasks, 400 million HEK293 EBNAcells were seeded 24 hours before transfection. For transfection cellswere centrifuged for 5 minutes at 210 g, and supernatant was replaced bypre-warmed CD CHO medium. Expression vectors were resuspended in 20 mLof CD CHO medium containing 200 pg of vector DNA. After addition of 540μL of polyethylenimine (PEI), the solution was mixed for 15 seconds andincubated for 10 minutes at room temperature. Afterwards, cells weremixed with the DNA/PEI solution, transferred to a 500 mL shake flask andincubated for 3 hours at 37° C. in an incubator with a 5% CO₂atmosphere. After the incubation, 160 mL of F17 medium was added andcells were cultured for 24 hours. One day after transfection, 1 mMvalproic acid and 7% Feed 1 (Lonza) were added to the culture. Theproduction medium was also supplemented with 100 μM biotin. After 7 daysof culturing, the cell supernatant was collected by spinning down cellsfor 15 min at 210 g. The solution was sterile filtered (0.22 μm filter),supplemented with sodium azide to a final concentration of 0.01% (w/v),and kept at 4° C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant was loadedon a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibratedwith 40 mL 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Unboundprotein was removed by washing with at least 10 column volumes of 20 mMsodium phosphate, 20 mM sodium citrate pH 7.5. The bound protein waseluted using a linear pH-gradient created over 20 column volumes of 20mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. Thecolumn was then washed with 10 column volumes of 20 mM sodium citrate,100 mM sodium chloride, 100 mM glycine, pH 3.0. pH of collectedfractions was adjusted by adding 1/10 (v/v) of 0.5 M sodium phosphate,pH 8.0. The protein was concentrated and filtered prior to loading on aHiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mMhistidine, 140 mM sodium chloride, pH 6.0.

The protein concentration was determined by measuring the opticaldensity (OD) at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity and molecularweight of the FolR1-Fc-fusion was analyzed by SDS capillaryelectrophoresis in the presence and absence of a reducing agentfollowing the manufacturer instructions (instrument Caliper LabChipGX,Perkin Elmer). The aggregate content of samples was analyzed using aTSKgel G3000 SW XL analytical size-exclusion column (Tosoh) equilibratedin 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, 0.02%(w/v) NaN3, pH 6.7 running buffer at 25° C.

Purified antigen-Fc-fusion proteins were analyzed by surface plasmonresonance assays using commercially available antibodies to confirmcorrect and natural conformation of the antigens (data not shown).

TABLE 1 Antigens produced for isolation,selection and counter selection of human FolR1 antibodies ECD AccessionSeq ID Antigen (aa) number Sequence No human 25-234 P15328RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWR 227 FolR1KNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRC IQMWFDPAQGNPNEEVARFYAAAM human17-230 P14207 TMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSP 228 FolR2WKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKERFLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSG RCIQMWFDSAQGNPNEEVARFYAAAMHVNhuman 24-243 P41439 SARARTDLLNVCMNAKHHKTQPSPEDELYGQCSPWKK 229 FolR3NACCTASTSQELHKDTSRLYNFNWDHCGKMEPTCKRHFIQDSCLYECSPNLGPWIRQVNQSWRKERILNVPLCKEDCERWWEDCRTSYTCKSNWHKGWNWTSGINECPAGALCSTFESYFPTPAALCEGLWSHSFKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFYAAAMNAGAPSRGIIDS murine 25-232 P35846TRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPWKTN 230 FolR1SCCSTNTSQEAHKDISYLYRFNWNHCGTMTSECKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERILDVPLCKEDCQQWWEDCQSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTSAALCEEIWSHSYKLSNYSRGSGRCIQ MWFDPAQGNPNEEVARFYAEAMS cynomolgus25-234 G7PR14 EAQTRTARARTELLNVCMNAKHHKEKPGPEDKLHEQC 231 FolR1RPWKKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCERWWEDCRTSYCKSNWHKGWNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYKVSNYSRGS GRCIQMWFDPAQGNPNEEVARFYAAAMS

TABLE 2 Summary of the yield and final monomer content of theFolR-Fc-fusions. Monomer [%] Antigen (SEC) Yield huFolR1 100 30 mg/LcyFolR1 100 32 mg/L muFolR1 100 31 mg/L huFolR2 100 16 mg/L huFolR3 9538 mg/L

Example 2 Generation of Common Light Chain with CD3ε Specificity

The T cell activating bispecific molecules described herein comprise atleast one CD3 binding moiety. This moiety can be generated by immunizinglaboratory animals, screening phage library or using known anti-CD3antibodies. The common light chain with CD3ε specificity was generatedby humanizing the light chain of a murine parental anti-CD3ε antibody(CH2527). For humanization of an antibody of non-human origin, the CDRresidues from the non-human antibody (donor) have to be transplantedonto the framework of a human (acceptor) antibody. Generally, acceptorframework sequences are selected by aligning the sequence of the donorto a collection of potential acceptor sequences and choosing one thathas either reasonable homology to the donor, or shows similar aminoacids at some positions critical for structure and activity. In thepresent case, the search for the antibody acceptor framework wasperformed by aligning the mouse VL-domain sequence of the parentalantibody to a collection of human germline sequences and choosing thehuman sequence that showed high sequence identity. Surprisingly, a goodmatch in terms of framework sequence homology was found in a ratherinfrequent human light chain belonging to the V-domain family 7 of thelambda type, more precisely, hVL_7_46 (IMGT nomenclature, GenBank AccNo. Z73674). This infrequent human light chain was subsequently chosenas acceptor framework for humanization of the light chain of CH2527. Thethree complementarity determining regions (CDRs) of the mouse lightchain variable domain were grafted onto this acceptor framework. Sincethe framework 4 region is not part of the variable region of thegermline V-gene, the alignment for this region (J-element) was doneindividually. Hence the IGU3-02 sequence was chosen for humanization ofthis light chain.

Thirteen humanized variants were generated (CH2527-VL7_46-1 toVL7_46-10, VL7_46-12 to VL7_46-14). These differ in framework residues(and combinations thereof) that were back-mutated to the murine V-domainsequence or in CDR-residues (Kabat definition) that could be keptidentical to the human germline sequence. The following frameworkresidues outside the CDRs were back-mutated to the murine residues inthe final humanized VL-domain variant VL7_46-13 (murine residueslisted): V36, E38, F44, G46, G49, and G57, respectively. The humanJ-element IGU3-02 was 100% identical to the J-element of the murineparental antibody.

Example 3 SPR Assessment of Humanized Variants with CD3ε Specificity

Humanized VL variants were assessed as chimera in a 2+1 TCB format, i.e.humanized light chain V-domains were paired with murine heavy chainV-domains. SPR assessment was carried out on a ProteOn XPR36 instrument(Bio-Rad). More precisely, the variants were captured directly from theculture supernatant on an anti-Fab derivatized GLM sensorchip (GoatAnti-Human IgG, F(ab′)₂ Fragment Specific, Jackson ImmunoResearch) invertical orientation. The following analytes were subsequently injectedhorizontally as single concentrations to assess binding to human andcynomolgus CD3ε: 3 μM hu CD3ε(−1-26)-Fc(knob)-avi (ID807) and 2.5 μM cyCD3ε-(-1-26)-Fc(knob)-Avi-Fc(hole) (ID873), respectively. Bindingresponses were qualitatively compared to binding of the murine controlconstruct and graded + (comparable binding observed), +/−(reducedbinding observed) and − (no binding observed). The capture antibody wasregenerated after each cycle of ligand capture and analyte binding andthe murine construct was re-injected at the end of the study to confirmthe activity of the capture surface. The results are summarized in Table3.

TABLE 3 Qualitative binding assessment based on SPR for the humanizedlight chain variants combined with the murine heavy chain of CH2527.Only the humanized light chain variant that was finally chosen,CH2527-VL7_46-13, highlighted in bold letters, exhibited comparablebinding to human and cynomolgus CD3ε. humanized VL variant binding toCD3ε murine_CH2527-VL + CH2527-VL7_46-1 − CH2527-VL7_46-2 −CH2527-VL7_46-3 − CH2527-VL7_46-4 − CH2527-VL7_46-5 − CH2527-VL7_46-6 −CH2527-VL7_46-7 − CH2527-VL7_46-8 − CH2527-VL7_46-9 − CH2527-VL7_46-10 −CH2527-VL7_46-12 +/− CH2527-VL7_46-13 + CH2527-VL7_46-14 −

Example 4 Properties of Humanized Common Light Chain with CD3εSpecificity

The light chain V-domain variant that was chosen for the humanized leadmolecule is VL7_46-13. The degree of humanness, i.e. the sequencehomology of the humanized V-domain to the human germline V-domainsequence was determined. For VL7_46-13, the overall sequence identitywith the closest human germline homolog is 65% before humanization and80% afterwards. Omitting the CDR regions, the sequence identity is 92%to the closest human germline homolog. As can be seen from Table 3,VL7_46-13 is the only humanized VL variant out of a panel of 13 variantsthat showed comparable binding to the parental murine antibody and alsoretained its cross-reactivity to cynomolgus CD3ε. This result indicatesthat it was not trivial to humanize the murine VL-domain without losingbinding affinity to CD3ε which required several back-mutations to murineframework residues (in particular G46) while retaining G24 in CDR1. Inaddition, this result shows that the VL-domain plays a crucial role intarget recognition. Importantly, the humanized VL-domain VL7_46-13 basedon an infrequent human germline belonging to the V-domain family 7 ofthe lambda type and retaining affinity and specificity for CD3ε, is alsosuitable to be used as a common light chain in phage-displayed antibodylibraries of the Fab-format and enables successful selection for novelspecificities which greatly facilitates the generation and production ofbispecific molecules binding to CD3ε and e.g. a tumor target and sharingthe same ‘common’ light chain.

Example 5 Generation of a Phage Displayed Antibody Library Using a HumanGerm-Line Common Light Chain Derived from HVK1-39

Several approaches to generate bispecific antibodies that resemble fulllength human IgG utilize modifications in the Fc region that induceheterodimerization of two distinct heavy chains. Such examples includeknobs-into-holes (Merchant et al., Nat Biotechnol. 1998 July;16(7):677-81) SEED (Davis et al., Protein Eng Des Sel. 2010 April;23(4):195-202) and electrostatic steering technologies (Gunasekaran etal., J Biol Chem. 2010 Jun. 18; 285(25):19637-46). Although theseapproaches enable effective heterodimerization of two distinct heavychains, appropriate pairing of cognate light and heavy chains remains aproblem. Usage of a common light chain (LC) can solve this issue(Merchant, et al. Nat Biotech 16, 677-681 (1998)).

Here, we describe the generation of an antibody library for the displayon a M13 phage. Essentially, we designed a multi framework library forthe heavy chain with one constant (or “common”) light chain. Thislibrary is designed for generating multispecific antibodies without theneed to use sophisticated technologies to avoid light chain mispairing.

By using a common light chain the production of these molecules can befacilitated as no mispairing occurs any longer and the isolation of ahighly pure bispecific antibody is facilitated. As compared to otherformats the use of Fab fragments as building blocks as opposed to e.g.the use of scFv fragments results in higher thermal stability and thelack of scFv aggregation and intermolecular scFv formation.

Library Generation

In the following the generation of an antibody library for the displayon M13 phage is described. Essentially, we designed a multi frameworklibrary for the heavy chain with one constant (or “common”) light chain.

We used these heavy chains in the library (GenBank Accession Numbers inbrackets):

IGHV1-46*01 (X92343) (SEQ ID NO:104),

IGHV1-69*06 (L22583), (SEQ ID NO:105)

IGHV3-15*01 (X92216), (SEQ ID NO:106)

IGHV3-23*01 (M99660), (SEQ ID NO:107)

IGHV4-59*01 (AB019438), (SEQ ID NO:108)

IGHV5-51*01 (M99686), (SEQ ID NO:109)

All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06which uses IGHJ6 sequence. The design of the randomization included theCDR-H1, CDR-H2, and CDR-H3. For CDR-H1 and CDR-H2 a “soft” randomizationstrategy was chosen, and the randomization oligonucleotides were suchthat the codon for the amino acid of the germ-line sequence was presentat 50%. All other amino acids, except cysteine, were summing up for theremaining 50%. In CDR-H3, where no germ-line amino acid is present dueto the presence of the genetic D-element, oligonucleotides were designedthat allow for the usage of randomized inserts between the V-element andthe J-element of 4 to 9 amino acids in length. Those oligonucleotidescontained in their randomized part e.g. The three amino acids G/Y/S arepresent to 15% each, those amino acids A/D/T/R/P/L/V/W/F/I/E are presentto 4.6% each.

Exemplary methods for generation of antibody libraries are described inHoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-413; Le et. al, J.Mol. Biol. (2004) 340, 1073-1093.

The light chain is derived from the human sequence hVK1-39, and is usedin an unmodified and non-randomized fashion. This will ensure that thesame light chain can be used for other projects without additionalmodifications.

Exemplary Library Selection:

Selections with all affinity maturation libraries are carried out insolution according to the following procedure using a monomeric andbiotinylated extracellular domain of a target antigen X.

1. 10{circumflex over ( )}12 phagemid particles of each library arebound to 100 nM biotinylated soluble antigen for 0.5 h in a total volumeof 1 ml. 2. Biotinylated antigen is captured and specifically boundphage particles are isolated by addition of ˜5×10{circumflex over ( )}7streptavidin-coated magnetic beads for 10 min. 3. Beads are washed using5-10×1 ml PBSTween20 and 5-10×1 ml PBS. 4. Elution of phage particles isdone by addition of 1 ml 100 mM TEA (triethylamine) for 10 min andneutralization by addition of 500 ul 1M Tris/HCl pH 7.4 and 5.Re-infection of exponentially growing E. coli TG1 bacteria, infectionwith helper phage VCSM13 and subsequent PEG/NaCl precipitation ofphagemid particles is applied in subsequent selection rounds. Selectionsare carried out over 3-5 rounds using either constant or decreasing(from 10{circumflex over ( )}-7M to 2×10{circumflex over ( )}-9M)antigen concentrations. In round 2, capture of antigen/phage complexesis performed using neutravidin plates instead of streptavidin beads. Allbinding reactions are supplemented either with 100 nM bovine serumalbumin, or with non-fat milk powder in order to compete for unwantedclones arising from mere sticky binding of the antibodies to the plasticsupport.

Selections are being carried out over three or four rounds usingdecreasing antigen concentrations of the antigen starting from 100 nMand going down to 5 nM in the final selection round. Specific bindersare defined as signals ca. 5× higher than background and are identifiedby ELISA. Specific binders are identified by ELISA as follows: 100 μl of10 nM biotinylated antigen per well are coated on neutravidin plates.Fab-containing bacterial supernatants are added and binding Fabs aredetected via their Flag-tags by using an anti-Flag/HRP secondaryantibody. ELISA-positive clones are bacterially expressed as soluble Fabfragments in 96-well format and supernatants are subjected to a kineticscreening experiment by SPR-analysis using ProteOn XPR36 (BioRad).Clones expressing Fabs with the highest affinity constants areidentified and the corresponding phagemids are sequenced. For furthercharacterization, the Fab sequences are amplified via PCR from thephagemid and cloned via appropriate restriction sites into human IgG1expression vectors for mammalian production.

Generation of a Phage Displayed Antibody Library Using a Humanized CD3εSpecific Common Light Chain

Here, the generation of an antibody library for the display on M13 phageis described. Essentially, we designed a multi framework library for theheavy chain with one constant (or “common”) light chain. This librarywas designed for the generation of Fc-containing, but FcgR bindinginactive T cell bispecific antibodies of IgG1 P329G LALA or IgG4 SPLE PGisotype in which one or two Fab recognize a tumor surface antigenexpressed on a tumor cell whereas the remaining Fab arm of the antibodyrecognizes CD3e on a T cell.

Library Generation

In the following the generation of an antibody library for the displayon M13 phage is described. Essentially, we designed a multi frameworklibrary for the heavy chain with one constant (or “common”) light chain.This library is designed solely for the generation of Fc-containing, butFcgR binding inactive T cell bispecific antibodies of IgG1 P329G LALA orIgG4 SPLE PG isotype.

Diversity was introduced via randomization oligonucleotides only in theCDR3 of the different heavy chains. Methods for generation of antibodylibraries are well known in the art and are described in (Hoogenboom etal., Nucleic Acids Res. 1991, 19, 4133-413; or in: Le et. al, J. Mol.Biol. (2004) 340, 1073-1093).

We used these heavy chains in the library:

IGHV1-46*01 (X92343), (SEQ ID NO:104)

IGHV1-69*06 (L22583), (SEQ ID NO:105)

IGHV3-15*01 (X92216), (SEQ ID NO:106)

IGHV3-23*01 (M99660), (SEQ ID NO:107)

IGHV4-59*01 (AB019438), (SEQ ID NO:108)

IGHV5-51*01 (M99686), (SEQ ID NO:109)

We used the light chain derived from the humanized human and CynomolgusCD3 ε specific antibody CH2527 in the library: (VL7_46-13; SEQ IDNO:112). This light chain was not randomized and used without anyfurther modifications in order to ensure compatibility with differentbispecific binders.

All heavy chains use the IGHJ2 as J-element, except the IGHV1-69*06which uses IGHJ6 sequence. The design of the randomization focused onthe CDR-H3 only, and PCR oligonucleotides were designed that allow forthe usage of randomized inserts between the V-element and the J-elementof 4 to 9 amino acids in length.

Example 6 Selection of Antibody Fragments from Common Light ChainLibraries (Comprising Light Chain with CD3ε Specificity) to FolR1

The antibodies 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 16D5, 15E12,21D1, 16F12, 21A5, 21G8, 19H3, 20G6, and 20H7 comprising the commonlight chain VL7_46-13 with CD3ε specificity were obtained by phagedisplay selections against different species (human, cynomolgus andmurine) of FolR1. Clones 16A3, 15A1, 18D3, 19E5, 19A4, 15H7, 15B6, 21D1,16F12, 19H3, 20G6, and 20H7 were selected from a sub-library in whichthe common light chain was paired with a heavy chain repertoire based onthe human germline VH1_46. In this sub-library, CDR3 of VH1_46 has beenrandomized based on 6 different CDR3 lengths. Clones 16D5, 15E12, 21A5,and 21G8 were selected from a sub-library in which the common lightchain was paired with a heavy chain repertoire based on the humangermline VH3_15. In this sub-library, CDR3 of VH3_15 has been randomizedbased on 6 different CDR3 lengths. In order to obtain speciescross-reactive (or murine FolR1-reactive) antibodies, the differentspecies of FolR1 were alternated (or kept constant) in different waysover 3 rounds of biopanning: 16A3 and 15A11 (human-cynomolgus-humanFolR1); 18D3 (cynomolgus-human-murine FolR1); 19E5 and 19A4 (3 roundsagainst murine FolR1); 15H7, 15B6, 16D5, 15E12, 21D1, 16F12, 21A5, 21G8(human-cynomolgus-human FolR1); 19H3, 20G6, and 20H7 (3 rounds againstmurine FolR1).

Human, murine and cynomolgus FolR1 as antigens for the phage displayselections as well as ELISA- and SPR-based screenings were transientlyexpressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and invivo site-specifically biotinylated via co-expression of BirA biotinligase at the avi-tag recognition sequence located at the C-terminus ofthe Fc portion carrying the receptor chain (Fc knob chain). In order toassess the specificity to FolR1, two related receptors, human FolR2 andFolR3 were generated in the same way.

Selection rounds (biopanning) were performed in solution according tothe following pattern:

1. Pre-clearing of ˜10¹² phagemid particles on maxisorp plates coatedwith 10 ug/ml of an unrelated human IgG to deplete the libraries ofantibodies recognizing the Fc-portion of the antigen.

2. Incubating the non-Fc-binding phagemid particles with 100 nMbiotinylated human, cynomolgus, or murine FolR1 for 0.5 h in thepresence of 100 nM unrelated non-biotinylated Fc knob-into-holeconstruct for further depletion of Fc-binders in a total volume of 1 ml.3. Capturing the biotinylated FolR1 and attached specifically bindingphage by transfer to 4 wells of a neutravidin pre-coated microtiterplate for 10 min (in rounds 1 & 3).4. Washing the respective wells using 5×PBS/Tween20 and 5×PBS.5. Eluting the phage particles by addition of 250 ul 100 mM TEA(triethylamine) per well for 10 min and neutralization by addition of500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells.6. Post-clearing of neutralized eluates by incubation on neutravidinpre-coated microtiter plate with 100 nM biotin-captured FolR2 or FolR3for final removal of Fc- and unspecific binders.7. Re-infection of log-phase E. coli TG1 cells with the supernatant ofeluted phage particles, infection with helperphage VCSM13, incubation ona shaker at 30° C. over night and subsequent PEG/NaCl precipitation ofphagemid particles to be used in the next selection round.

Selections were carried out over 3 rounds using constant antigenconcentrations of 100 nM. In round 2, in order to avoid enrichment ofbinders to neutravidin, capture of antigen: phage complexes wasperformed by addition of 5.4×10⁷ streptavidin-coated magnetic beads.Specific binders were identified by ELISA as follows: 100 ul of 25 nMbiotinylated human, cynomolgus, or murine FolR1 and 10 ug/ml of humanIgG were coated on neutravidin plates and maxisorp plates, respectively.Fab-containing bacterial supernatants were added and binding Fabs weredetected via their Flag-tags using an anti-Flag/HRP secondary antibody.Clones exhibiting signals on human FolR1 and being negative on human IgGwere short-listed for further analyses and were also tested in a similarfashion against the remaining two species of FolR. They were bacteriallyexpressed in a 0.5 liter culture volume, affinity purified and furthercharacterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor.

Affinities (K_(D)) of selected clones were measured by surface plasmonresonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated human, cynomolgus, and murine FolR1 as well as human FolR2and FolR3 (negative controls) immobilized on NLC chips by neutravidincapture. Immobilization of antigens (ligand): Recombinant antigens werediluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4,0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute in verticalorientation.

Injection of analytes: For ‘one-shot kinetics’ measurements, injectiondirection was changed to horizontal orientation, two-fold dilutionseries of purified Fab (varying concentration ranges) were injectedsimultaneously along separate channels 1-5, with association times of200 s, and dissociation times of 600 s. Buffer (PBST) was injected alongthe sixth channel to provide an “in-line” blank for referencing.Association rate constants (k_(on)) and dissociation rate constants(k_(off)) were calculated using a simple one-to-one Langmuir bindingmodel in ProteOn Manager v3.1 software by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (K_(D)) was calculated as the ratio k_(off)/k_(on). Table 4lists the equilibrium dissociation constants (K_(D)) of the selectedclones specific for FolR1.

TABLE 4 Equilibrium dissociation constants (KD) for anti-FolR1antibodies (Fab-format) selected by phage display from common lightchain sub-libraries comprising VL7_46-13, a humanized light chainspecific for CD3ε. KD in nM. huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3Clone [nM] [nM] [nM] [nM] [nM] 16A3 21.7 18 very weak no binding nobinding 15A1 30.9 17.3 very weak no binding no binding 18D3 93.6 40.2very weak no binding no binding 19E5 522 276 19.4 no binding no binding19A4 2050 4250 43.1 no binding no binding 15H7 13.4 72.5 no binding nobinding no binding 15B6 19.1 13.9 no binding no binding no binding 16D539.5 114 no binding no binding no binding 15E12 55.7 137 no binding nobinding no binding 21D1 62.6 32.1 no binding no binding no binding 16F1268 90.9 no binding no binding no binding 21A5 68.8 131 no binding nobinding no binding 21G8 130 261 no binding no binding no binding 19H3 nobinding no binding 89.7 no binding no binding 20G6 no binding no binding78.5 no binding no binding

Example 7 Selection of Antibody Fragments from Generic Multi-FrameworkLibraries to FolR1

The antibodies 11F8, 36F2, 9D11, 5D9, 6B6, and 14E4 were obtained byphage display selections based on generic multi-framework sub-librariesagainst different species (human, cynomolgus and murine) of FolR. Inthese multi-framework sub-libraries, different VL-domains withrandomized CDR3 (3 different lengths) are paired with differentVH-domains with randomized CDR3 (6 different lengths). The selectedclones are of the following VL/VH pairings: 11F8 (Vk_1_5/VH_1_69), 36F2(Vk_3_20/VH_1_46), 9D11 (Vk2D_28/VH1_46), 5D9 (Vk3_20/VH1_46), 6B6(Vk3_20/VH1_46), and 14E4 (Vk3_20/VH3_23). In order to obtain speciescross-reactive (or murine FolR1-reactive) antibodies, the differentspecies of FolR1 were alternated (or kept constant) in different waysover 3 or 4 rounds of biopanning: 11F8 (cynomolgus-murine-human FolR1);36F2 (human-murine-cynomolgus-murine FolR1); 9D11(cynomolgus-human-cynomolgus FolR1); 5D9 (human-cynomolgus-human FolR1);6B6 (human-cynomolgus-human FolR1) and 14E4 (3 rounds against murineFolR1).

Human, murine and cynomolgus FolR1 as antigens for the phage displayselections as well as ELISA- and SPR-based screenings were transientlyexpressed as N-terminal monomeric Fc-fusion in HEK EBNA cells and invivo site-specifically biotinylated via co-expression of BirA biotinligase at the avi-tag recognition sequence located at the C-terminus ofthe Fc portion carrying the receptor chain (Fc knob chain). In order toassess the specificity to FolR1, two related receptors, human FolR2 andFolR3 were generated in the same way.

Selection rounds (biopanning) were performed in solution according tothe following pattern:

1. Pre-clearing of ˜10¹² phagemid particles on maxisorp plates coatedwith 10 ug/ml of an unrelated human IgG to deplete the libraries ofantibodies recognizing the Fc-portion of the antigen.

2. Incubating the non-Fc-binding phagemid particles with 100 nMbiotinylated human, cynomolgus, or murine FolR1 for 0.5 h in thepresence of 100 nM unrelated non-biotinylated Fc knob-into-holeconstruct for further depletion of Fc-binders in a total volume of 1 ml.3. Capturing the biotinylated FolR1 and attached specifically bindingphage by transfer to 4 wells of a neutravidin pre-coated microtiterplate for 10 min (in rounds 1 & 3).4. Washing the respective wells using 5×PBS/Tween20 and 5×PBS.5. Eluting the phage particles by addition of 250 ul 100 mM TEA(triethylamine) per well for 10 min and neutralization by addition of500 ul 1 M Tris/HCl pH 7.4 to the pooled eluates from 4 wells.6. Post-clearing of neutralized eluates by incubation on neutravidinpre-coated microtiter plate with 100 nM biotin-captured FolR2 or FolR3for final removal of Fc- and unspecific binders.7. Re-infection of log-phase E. coli TG1 cells with the supernatant ofeluted phage particles, infection with helperphage VCSM13, incubation ona shaker at 30° C. over night and subsequent PEG/NaCl precipitation ofphagemid particles to be used in the next selection round.

Selections were carried out over 3 rounds using constant antigenconcentrations of 100 nM. In round 2 and 4, in order to avoid enrichmentof binders to neutravidin, capture of antigen: phage complexes wasperformed by addition of 5.4×10⁷ streptavidin-coated magnetic beads.Specific binders were identified by ELISA as follows: 100 ul of 25 nMbiotinylated human, cynomolgus, or murine FolR1 and 10 ug/ml of humanIgG were coated on neutravidin plates and maxisorp plates, respectively.Fab-containing bacterial supernatants were added and binding Fabs weredetected via their Flag-tags using an anti-Flag/HRP secondary antibody.Clones exhibiting signals on human FolR1 and being negative on human IgGwere short-listed for further analyses and were also tested in a similarfashion against the remaining two species of FolR. They were bacteriallyexpressed in a 0.5 liter culture volume, affinity purified and furthercharacterized by SPR-analysis using BioRad's ProteOn XPR36 biosensor.

Affinities (K_(D)) of selected clones were measured by surface plasmonresonance (SPR) using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated human, cynomolgus, and murine FolR1 as well as human FolR2and FolR3 (negative controls) immobilized on NLC chips by neutravidincapture. Immobilization of antigens (ligand): Recombinant antigens werediluted with PBST (10 mM phosphate, 150 mM sodium chloride pH 7.4,0.005% Tween 20) to 10 μg/ml, then injected at 30 μl/minute in verticalorientation. Injection of analytes: For ‘one-shot kinetics’measurements, injection direction was changed to horizontal orientation,two-fold dilution series of purified Fab (varying concentration ranges)were injected simultaneously along separate channels 1-5, withassociation times of 150 or 200 s, and dissociation times of 200 or 600s, respectively. Buffer (PBST) was injected along the sixth channel toprovide an “in-line” blank for referencing. Association rate constants(k_(on)) and dissociation rate constants (k_(off)) were calculated usinga simple one-to-one Langmuir binding model in ProteOn Manager v3.1software by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) wascalculated as the ratio k_(off)/k_(on). Table 5 lists the equilibriumdissociation constants (K_(D)) of the selected clones specific forFolR1.

TABLE 5 Equilibrium dissociation constants (K_(D)) for anti-FolR1antibodies (Fab-format) selected by phage display from generic multi-framework sub-libraries. K_(D) in nM. K_(D) (nM) Clone huFolR1 cyFolR1muFolR1 huFolR2 huFolR3 11F8 632 794 1200 no binding no binding 36F21810 1640 737 no binding no binding 9D11 8.64 5.29 no binding no bindingno binding 5D9 8.6 5.9 no binding no binding no binding 6B6 14.5 9.4 nobinding no binding no binding 14E4 no binding no binding 6.09 no bindingno binding.

Example 8 Production and Purification of Novel FolR1 Binders in IgG andT-Cell Bispecific Formats

To identify FolR1 binders which are able to induce T-cell dependentkilling of selected target cells the antibodies isolated from a commonlight chain- or Fab-library were converted into the corresponding humanIgG1 format. In brief, the variable heavy and variable light chains ofunique FolR1 binders from phage display were amplified by standard PCRreactions using the Fab clones as the template. The PCR products werepurified and inserted (either by restriction endonuclease and ligasebased cloning, or by ‘recombineering’ using the InFusion kit fromInvitrogen) into suitable expression vectors in which they are fused tothe appropriate human constant heavy or human constant light chain. Theexpression cassettes in these vectors consist of a chimeric MPSVpromoter and a synthetic polyadenylation site. In addition, the plasmidscontain the oriP region from the Epstein Barr virus for the stablemaintenance of the plasmids in HEK293 cells harboring the EBV nuclearantigen (EBNA). After PEI mediated transfection the antibodies weretransiently produced in HEK293 EBNA cells and purified by standardProteinA affinity chromatography followed by size exclusionchromatography as described:

Transient Transfection and Production

All (bispecific) antibodies (if not obtained from a commercial source)used herein were transiently produced in HEK293 EBNA cells using a PEImediated transfection procedure for the required vectors as describedbelow. HEK293 EBNA cells are cultivated in suspension serum free in CDCHO culture medium. For the production in 500 ml shake flask 400 millionHEK293 EBNA cells are seeded 24 hours before transfection (foralternative scales all amounts were adjusted accordingly). Fortransfection cells are centrifuged for 5 min by 210×g, supernatant isreplaced by pre-warmed 20 ml CD CHO medium. Expression vectors are mixedin 20 ml CD CHO medium to a final amount of 200 μg DNA. After additionof 540 μl PEI solution is vortexed for 15 s and subsequently incubatedfor 10 min at room temperature. Afterwards cells are mixed with theDNA/PEI solution, transferred to a 500 ml shake flask and incubated for3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. Afterincubation time 160 ml F17 medium is added and cell are cultivated for24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 isadded. After 7 days cultivation supernatant is collected forpurification by centrifugation for 15 min at 210×g, the solution issterile filtered (0.22 μm filter) and sodium azide in a finalconcentration of 0.01% w/v is added, and kept at 4° C. After productionthe supernatants were harvested and the antibody containing supernatantswere filtered through 0.22 μm sterile filters and stored at 4° C. untilpurification.

Antibody Purification

All molecules were purified in two steps using standard procedures, suchas protein A affinity purification (Äkta Explorer) and size exclusionchromatography. The supernatant obtained from transient production wasadjusted to pH 8.0 (using 2 M TRIS pH 8.0) and applied to HiTrap PA FF(GE Healthcare, column volume (cv)=5 ml) equilibrated with 8 columnvolumes (cv) buffer A (20 mM sodium phosphate, 20 mM sodium citrate, pH7.5). After washing with 10 cv of buffer A, the protein was eluted usinga pH gradient to buffer B (20 mM sodium citrate pH 3, 100 mM NaCl, 100mM glycine) over 12 cv. Fractions containing the protein of interestwere pooled and the pH of the solution was gently adjusted to pH 6.0(using 0.5 M Na₂HPO₄ pH 8.0). Samples were concentrated to 2 ml usingultra-concentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius) andsubsequently applied to a HiLoad™ 16/60 Superdex™ 200 preparative grade(GE Healthcare) equilibrated with 20 mM Histidine, pH 6.0, 140 mM NaCl,0.01% Tween-20. The aggregate content of eluted fractions was analyzedby analytical size exclusion chromatography. Therefore, 30 μl of eachfraction was applied to a TSKgel G3000 SW XL analytical size-exclusioncolumn (Tosoh) equilibrated in 25 mM K₂HPO₄, 125 mM NaCl, 200 mML-arginine monohydrochloride, 0.02% (w/v) NaN₃, pH 6.7 running buffer at25° C. Fractions containing less than 2% oligomers were pooled andconcentrated to final concentration of 1-1.5 mg/ml using ultraconcentrators (Vivaspin 15R 30.000 MWCO HY, Sartorius). The proteinconcentration was determined by measuring the optical density (OD) at280 nm, using the molar extinction coefficient calculated on the basisof the amino acid sequence. Purity and molecular weight of theconstructs were analyzed by SDS capillary electrophoresis in thepresence and absence of a reducing agent following the manufacturerinstructions (instrument Caliper LabChipGX, Perkin Elmer). Purifiedproteins were frozen in liquid N2 and stored at −80° C.

Based on in vitro characterization results selected binders we convertedinto a T-cell bispecific format. In these molecules the FolR1:CD3binding moieties are arranged in a 2:1 order with the FolR11 Fabs beinglocated at the N-terminus. For clones isolated from the standard Fablibrary the CD3 binding part was generated as a CrossFab (CH1Cκcrossing) while for the clones from the common light chain library nocrossing was necessary. These bispecific molecules were produced andpurified analogously to the IgGs.

TABLE 6 Yield and monomer content of novel FolR1 binders in IgG and TCBformat, respectively. IgG TCB Yield Monomer Yield Monomer # CloneLibrary [mg/L] [%] [mg/L} [%]  1 11F8 Fab 8.03 96.26 — —  2 14E4 Fab8.90 98.12 — —  3 15B6 CLC 7.72 100.00 — —  4 15E12 CLC 6.19 100.00 — — 5 15H7 CLC 8.94 100.00 — —  6 16A3 CLC 0.60 n.d. — —  7 16D5 CLC 36.5096.96 4.36 97.19  8 16F12 CLC 5.73 97.17 — —  9 18D3 CLC 0.90 n.d. — —10 19A4 CLC 38.32 100.00 37.50 100.00 11 19E5 CLC 46.09 100.00 — — 1219H3 CLC 7.64 100.00 — — 13 20G6 CLC 24.00 100.00 — — 14 20H7 CLC 45.39100.00 — — 15 21A5 CLC 1.38 98.56 47.31 95.08 16 21D1 CLC 5.47 100.00 —— 17 21G8 CLC 6.14 97.28 9.27 100.00 18 36F2 Fab 11.22 100.00 18.00100.00 19 5D9 Fab 20.50 100.00 0.93 97.32 20 6B6 Fab 3.83 100.00 4.1791.53 21 9D11 Fab 14.61 100.00 2.63 100.00 CLC: Common light chain

Example 9 2+1 and 1+1 T-Cell Bispecific Formats

Four different T-cell bispecific formats were prepared for one commonlight chain binder (16D5) and three formats for one binder from the Fablibrary (9D11) to compare their killing properties in vitro.

The standard format is the 2+1 inverted format as already described(FolR1:CD3 binding moieties arranged in a 2:1 order with the FolR1 Fabslocated at the N-terminus). In the 2+1 classical format the FolR1:CD3binding moieties are arranged in a 2:1 order with the CD3 Fab beinglocated at the N-terminus. Two monovalent formats were also prepared.The 1+1 head-to-tail has the FolR1:CD3 binding moieties arranged in a1:1 order on the same arm of the molecule with the FolR1 Fab located atthe N-terminus. In the 1+1 classical format the FolR1:CD3 bindingmoieties are present once, each on one arm of the molecule. For the 9D11clone isolated from the standard Fab library the CD3 binding part wasgenerated as a CrossFab (CH1Cκ crossing) while for the 16D5 from thecommon light chain library no crossing was necessary. These bispecificmolecules were produced and purified analogously to the standardinverted T-cell bispecific format.

TABLE 7 Summary of the yield and final monomer content of the differentT-cell bispecific formats. Monomer [%] Construct (SEC) Yield 16D5 FolR1TCB 2 + 1 (inverted)  96% 5.4 mg/L 16D5 FolR1 TCB 2 + 1 (classical)  90%4.6 mg/L 16D5 FolR1 TCB 1 + 1 (head-to- 100 % 5.4 mg/L tail) 16D5 FolR1TCB 1 + 1 (classical) 100% 0.7 mg/L 9D11 FolR1 TCB 2 + 1 (inverted) 100%2.6 mg/L 9D11 FolR1 TCB 1 + 1 (head-to- 100% 6.1 mg/L tail) 9D11 FolR1TCB 1 + 1 (classical)  96% 1.3 mg/L Mov19 FolR1 TCB 2 + 1 (inverted) 98% 3 mg/L Mov19 FolR1 TCB 1 + 1 (head-to- 100% 5.2 mg/L tail)

Example 10 Biochemical Characterization of FolR1 Binders by SurfacePlasmon Resonance

Binding of FolR1 binders as IgG or in the T-cell bispecific format todifferent recombinant folate receptors (human FolR1, 2 and 3, murineFolR1 and cynomolgus FolR1; all as Fc fusions) was assessed by surfaceplasmon resonance (SPR). All SPR experiments were performed on a BiacoreT200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

Single Injections

First the anti-FolR1 IgGs were analyzed by single injections (Table 1)to characterize their crossreactivity (to human, murine and cyno FolR1)and specificity (to human FolR1, human FolR2, human FolR3). Recombinantbiotinylated monomeric Fc fusions of human, cynomolgus and murine FolateReceptor 1 (FolR1-Fc) or human Folate Receptor 2 and 3 (FolR2-Fc,FolR3-Fc) were directly coupled on a SA chip using the standard couplinginstruction (Biacore, Freiburg/Germany). The immobilization level wasabout 300-400 RU. The IgGs were injected for 60 seconds at aconcentration of 500 nM. IgGs binding to huFolR2 and huFolR3 wererejected for lack of specificity. Most of the binders are onlycrossreactive between human and cyno FolR1, additional crossreactivityto murine FolR1 went most of the time hand in hand with loss ofspecificity.

TABLE 8 Crossreactivity and specificity of 25 new folate receptor 1binders (as IgGs) as well as of two control IgGs (Mov19 andFarletuzumab). + means binding, − means no binding, +/− means weakbinding. Binding to Binding to Binding to Binding to Binding to Clonename huFolR1 cyFolR1 muFolR1 huFolR2 huFolR3 Mov19 + + − − −Farletuzumab + + − − − 16A3 + + +/− − − 18D3 + + − − − 19E5 + + + + +19A4 − − + + + 15H7 + + + − − 15B6 + + − − − 16D5 + + − − − 15E12 + ++/− + + 21D1 + + +/− − − 16F12 + + − − − 21A5 + + − − +/− 21G8 + + − + +19H3 − − + − − 20G6 − − + − − 20H7 − − + − − 9D11 + + − − − 5D9 + +− + + 6B6 + + − + + 11F8 + + + + + 36F2 + + + − − 14E4 − − + − −Avidity to Folate Receptor 1

The avidity of the interaction between the anti-FolR1 IgGs or T cellbispecifics and the recombinant folate receptors was determined asdescribed below (Table 9).

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus andmurine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chipusing the standard coupling instruction (Biacore, Freiburg/Germany). Theimmobilization level was about 300-400 RU. The anti-FolR1 IgGs or T cellbispecifics were passed at a concentration range from 2.1 to 500 nM witha flow of 30 μL/minutes through the flow cells over 180 seconds. Thedissociation was monitored for 600 seconds. Bulk refractive indexdifferences were corrected for by subtracting the response obtained onreference flow cell immobilized with recombinant biotinylated IL2receptor Fc fusion. For the analysis of the interaction of 19H3 IgG andmurine folate receptor 1, folate (Sigma F7876) was added in the HBS-EPrunning buffer at a concentration of 2.3 μM. The binding curvesresulting from the bivalent binding of the IgGs or T cell bispecificswere approximated to a 1:1 Langmuir binding and fitted with that model(which is not correct, but gives an idea of the avidity). The apparentavidity constants for the interactions were derived from the rateconstants of the fitting using the Bia Evaluation software (GEHealthcare).

TABLE 9 Bivalent binding (avidity with apparent KD) of selected FolR1binders as IgGs or as T-cell bispecifics (TCB) on human and cyno FolRl.Apparent Analyte Ligand ka (1/Ms) kd (Vs) KD (M) 16D5 TCB huFolR18.31E+04 3.53E−04 4.24E−09 cyFolR1 1.07E+05 3.70E−04 3.45E−09 9D11 TCBhuFolR1 1.83E+05 9.83E−05 5.36E−10 cyFolR1 2.90E+05 6.80E−05 2.35E−1021A5 TCB huFolR1 2.43E+05 2.64E−04 1.09E−09 cyFolR1 2.96E+05 2.76E−049.32E−10 36F2 IgG huFolR1 2.62E+06 1.51E−02 5.74E−9  cyFolR1 3.02E+061.60E−02 5.31E−9  muFolR1  3.7E+05 6.03E−04 1.63E−9  Mov19 IgG huFolR18.61E+05 1.21E−04  1.4E−10 cyFolR1 1.29E+06 1.39E−04 1.08E−10Farletuzumab huFolR1 1.23E+06    9E−04  7.3E−10 cyFolR1 1.33E+068.68E−04  6.5E−10 19H3 IgG muFolR1  7.1E+05  1.1E−03 1.55E−091. Affinity to Folate Receptor 1

The affinity of the interaction between the anti-FolR1 IgGs or the Tcell bispecifics and the recombinant folate receptors was determined asdescribed below (Table 10).

For affinity measurement, direct coupling of around 6000-7000 resonanceunits (RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 IgGs or T cellbispecifics were captured at 20 nM with a flow rate of 10 μl/min for 20or 40 sec, the reference flow cell was left without capture. Dilutionseries (6.17 to 500 nM or 12.35 to 3000 nM) of human or cyno FolateReceptor 1Fc fusion were passed on all flow cells at 30 μl/min for 120or 240 sec to record the association phase. The dissociation phase wasmonitored for 240 sand triggered by switching from the sample solutionto HBS-EP. The chip surface was regenerated after every cycle using adouble injection of 60 sec 10 mM Glycine-HCl pH 2.1 or pH1.5. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on the reference flow cell 1. The affinity constantsfor the interactions were derived from the rate constants by fitting toa 1:1 Langmuir binding using the Bia Evaluation software (GEHealthcare).

TABLE 10 Monovalent binding (affinity) of selected FolR1 binders as IgGsor as T-cell bispecifics (TCB) on human and cyno FolRl. Ligand Analyteka (1/Ms) kd (1/s) KD (M) 16D5 TCB huFolR1 1.53E+04 6.88E−04 4.49E−08cyFolR1 1.32E+04 1.59E−03 1.21E−07 9D11 TCB huFolR1 3.69E+04 3.00E−048.13E−09 cyFolR1 3.54E+04 2.06E−04 5.82E−09 21A5 TCB huFolR1 1.79E+04 1.1E−03 6.16E−08 cyFolR1 1.48E+04 2.06E−03  1.4E−07 Mov19 IgG huFolR12.89E+05 1.59E−04  5.5E−10 cyFolR1 2.97E+05 1.93E−04  6.5E−10Farletuzumab huFolR1 4.17E+05 2.30E−02 5.53E−08 cyFolR1 5.53E+053.73E−02 6.73E−082. Affinity to CD3

The affinity of the interaction between the anti-FolR1 T cellbispecifics and the recombinant human CD3εδ-Fc was determined asdescribed below (Table 11).

For affinity measurement, direct coupling of around 9000 resonance units(RU) of the anti-human Fab specific antibody (Fab capture kit, GEHealthcare) was performed on a CM5 chip at pH 5.0 using the standardamine coupling kit (GE Healthcare). Anti-FolR1 T cell bispecifics werecaptured at 20 nM with a flow rate of 10 μl/min for 40 sec, thereference flow cell was left without capture. Dilution series (6.17 to500 nM) of human CD3εδ-Fc fusion were passed on all flow cells at 30μl/min for 240 sec to record the association phase. The dissociationphase was monitored for 240 s and triggered by switching from the samplesolution to HBS-EP. The chip surface was regenerated after every cycleusing a double injection of 60 sec 10 mM Glycine-HCl pH 2.1. Bulkrefractive index differences were corrected for by subtracting theresponse obtained on the reference flow cell 1. The affinity constantsfor the interactions were derived from the rate constants by fitting toa 1:1 Langmuir binding using the Bia Evaluation software (GEHealthcare).

TABLE 11 Monovalent binding (affinity) of selected FolR1 T-cellbispecifics (TCB) on human CD3-Fc. Ligand Analyte ka (1/Ms) kd (1/s) KD(M) 16D5 TCB huCD3 4.25E+04 3.46E−03 8.14E−08 21A5 TCB huCD3 3.72E+043.29E−03  8.8E−08

The CD3 binding part is identical for all constructs and the affinity issimilar for the measured T cell bispecifics (K_(D) range between 60 and90 nM).

Example 11 Simultaneous Binding T Cell Bispecifics on Folate Receptor 1and CD3

Simultaneous binding of the anti-FolR1 T cell bispecifics on recombinantFolate Receptor 1 and recombinant human CD3εδ-Fc was determined asdescribed below.

Recombinant biotinylated monomeric Fc fusions of human, cynomolgus andmurine Folate Receptor 1 (FolR1-Fc) were directly coupled on a SA chipusing the standard coupling instruction (Biacore, Freiburg/Germany). Theimmobilization level was about 300-400 RU. The anti-FolR1 T cellbispecifics were injected for 60 s at 500 nM with a flow of 30μl/minutes through the flow cells, followed by an injection of huCDεδ-Fc for 60 s at 500 nM. Bulk refractive index differences werecorrected for by subtracting the response obtained on reference flowcell immobilized with recombinant biotinylated IL2 receptor Fc fusion.The four T cell bispecifics tested (16D5 TCB, 21A5 TCB, 51C7 TCB and45D2 TCB) were able to bind simultaneously to Folate Receptor 1 andhuman CD3 as expected.

Example 12 Epitope Binning

For epitope binning, the anti-FolR1 IgGs or T cell bispecifics weredirectly immobilized on a CM5 chip at pH 5.0 using the standard aminecoupling kit (GE Healthcare), with a final response around 700 RU. 500nM huFolR1-Fc was then captured for 60 s, followed by 500 nM of thedifferent binders for 30 s. The surface was regenerated with twoinjections of 10 mM glycine pH 2 for 30 s each. It is assessed if thedifferent binders can bind to huFolR1 captured on immobilized binders(Table 12).

TABLE 12 Epitope characterization of selected FolR1 binders as IgGs oras T-cell bispecifics (TCB) on human FolR1. +means binding, −means nobinding, +/−means weak binding Analytes in solution On 16D5 21A5 9D1136F2 Mov19 huFolR1 TCB TCB TCB IgG IgG Farletuzumab Immobilized 16D5 TCB− − − + + + 21A5 TCB − − − + + + 9D11 TCB No additional binding on FolR1possible once captured on 9D11 36F2 IgG Measure not possible, huFolR1dissociates too rapidly Mov19 IgG + + +/− − − −

Based on these results and additional data with simultaneous binding onimmobilized huFolR1, the binders were separated in three groups. Itisnot clear if 9D11 has a separate epitope because it displaces all theother binders. 16D5 and 21A5 seem to be in the same group and Mov9,Farletuzumab (Coney et al., Cancer Res. 1991 Nov. 15; 51(22):6125-32;Kalli et al., Curr Opin Investig Drugs. 2007 December; 8(12):1067-73)and 36F2 in another (Table 13). However, 36F2 binds to a differentepitope than Mov 19 and Farletuzumab as it binds to human, cynomous andmurine FolR1.

TABLE 13 Epitope grouping of selected FolR1 binders as IgGs or as T-cellbispecifics (TCB) on human FolR1 Epitope 1 Epitope 2 Epitope 3 16D5 9D11Mov19 21A5 Farletuzumab 36F2

Example 13 Selection of Binders

FolR1 binders in the IgG formats were screened by surface plasmonresonance (SPR) and by in vitro assay on cells to select the bestcandidates.

The anti-FolR1 IgGs were analyzed by SPR to characterize theircrossreactivity (to human, murine and cynomolgus FolR1) and specificity(to human FolR1, human FolR2, human FolR3). Unspecific binding to humanFolR2 and 3 was considered an exclusion factor. Binding and specificityto human FolR1 was confirmed on cells. Some binders did not bind oncells expressing FolR1 even though they recognized the recombinant humanFolR1 in SPR. Aggregation temperature was determined but was not anexclusion factor because the selected binders were all stable. Selectedbinders were tested in a polyreactivity ELISA to check for unspecificbinding, which led to the exclusion of four binders. This processresulted in an initial selection of three binders: 36F2 (Fab library),9D11 (Fab library) and 16D5 (common light chain). 36F2 dissociatedrapidly from huFolR1 and was, therefore, initially not favored.

Example 14 Specific Binding of Newly Generated FolR1 Binders to HumanFolR1 Positive Tumor Cells

New FolR1 binders were generated via Phage Display using either a Fablibrary or a common light chain library using the CD3 light chain. Theidentified binders were converted into a human IgG1 format and bindingto FolR1 high expressing HeLa cells was addressed. As reference moleculethe human FolR1 binder Mov19 was included. Most of the binders tested inthis assay showed intermediate to good binding to FolR1 with some clonesbinding equally well as Mov19 (see FIG. 2 ). The clones 16A3, 18D3,15H7, 15B6, 21D1, 14E4 and 16F12 were excluded because binding to FolR1on cells could not be confirmed by flow cytometry. In a next step theselected clones were tested for specificity to human FolR1 by excludingbinding to the closely related human FolR2. HEK cells were transientlytransfected with either human FolR1 or human FolR2 to addressspecificity. The clones 36F2 and 9D11 derived from the Fab library andthe clones 16D5 and 21A5 derived from the CLC library bind specificallyto human FolR1 and not to human FolR2 (see FIGS. 3A-B). All the othertested clones showed at least some binding to human FolR2 (see FIGS.3A-B). Therefore these clones were excluded from furthercharacterization. In parallel cross-reactivity of the FolR1 clones tocyno FolR1 was addressed by performing binding studies to HEK cellstransiently transfected with cyno FolR1. All tested clones were able tobind cyno FolR1 and the four selected human FoLR1 specific clones 36F2,9D11, 16D5 and 21A5 bind comparably well human and cyno FoLR1 (FIG. 4 ).Subsequently three human FolR1 specific cyno cross-reactive binders wereconverted into TCB format and tested for induction of T cell killing andT cell activation. These clones were 9D11 from the Fab library and 16D5and 21A5 from the CLC library. As reference molecule Mov19 FolR1 TCB wasincluded in all studies. These FolR1 TCBs were then used to compareinduction of internalization after binding to FolR1 on HeLa cells. Allthree tested clones are internalized upon binding to FolR1 comparable tointernalization upon binding of Mov19 FoLR1 TCB (FIG. 5 ). 21A5 FolR1TCB was discontinued due to signs of polyreactivity.

Example 15 T Cell-Mediated Killing of FolR1-Expressing Tumor TargetCells Induced by FolR1 TCB Antibodies

The FolR1 TCBs were used to determine T cell mediated killing of tumorcells expressing FoLR1. A panel of potential target cell lines was usedto determine FoLR1 binding sites by Qifikit analysis.

The used panel of tumor cells contains FolR1 high, intermediate and lowexpressing tumor cells and a FolR1 negative cell line.

TABLE 14 FolR1 binding sites on tumor cells Cell line Origin FolR1binding sites Hela Cervix adenocarcinoma 2'240'716 Skov3 Ovarianadenocarcinoma 91'510 OVCAR5 Ovarian adenocarcinoma 22'077 HT29Colorectal adenocarcinoma 10'135 MKN45 Gastric adenocarcinoma 54

Binding of the three different FoLR1 TCBs (containing 9D11, 16D5 andMov19 binders) to this panel of tumor cell lines was determined showingthat the FoLR1 TCBs bind specifically to FolR1 expressing tumor cellsand not to a FoLR1 negative tumor cell line. The amount of boundconstruct is proportional to the FolR1 expression level and there isstill good binding of the constructs to the FolR1 low cell line HT-29detectable. In addition there is no binding of the negative control DP47TCB to any of the used cell lines (FIGS. 6A-E). DP47 TCB is anuntargeted TCB and was prepared as described in WO2014/131712.

The intermediate expressing cell line SKOV3 and the low expressing cellline HT-29 were further on used to test T cell mediated killing and Tcell activation using 16D5 TCB and 9D11 TCB; DP47 TCB was included asnegative control. Both cell lines were killed in the presence of alreadyvery low levels of 16D5 TCB and 9D11 TCB and there was no difference inactivity between both TCBs even though 9D11 TCB binds stronger to FolR1than 16D5 TCB. Overall killing of SKOV3 cells was higher compared toHT-29 which reflects the higher expression levels of FolR1 on SKOV3cells (FIGS. 7A-D). In line with this, a strong upregulation of theactivation marker CD25 and CD69 on CD4⁺ T cells and CD8⁺ T cells wasdetected. Activation of T cells was very similar in the presence ofSKOV3 cells and HT-29 cells. The negative control DP47 TCB does notinduce any killing at the used concentrations and there was nosignificant upregulation of CD25 and CD69 on T cells.

TABLE 15 EC50 values of tumor cell killing and T cell activation withSKOV3 cells Killing Killing CD4 + CD4 + CD8 + CD8 + 24 h 48 h CD69 +CD25 + CD69 + CD25 + Construct (pM) (pM) (%) (%) (%) (%) 9D11 1.1 0.030.51 0.46 0.019 0.03  FolR1 TCB 16D5 0.7 0.04 0.34 0.33 0.025 0.031FolR1 TCB

TABLE 16 EC50 values of tumor cell killing and T cell activation withHT-29 cells CD4 + CD4 + CD8 + CD8 + Killing Killing CD69 + CD25 + CD69 +CD25 + Construct 24 h (pM) 48 h (pM) (%) (%) (%) (%) 9D11 2.3 0.1 1.221.11 0.071 0.084 FolR1 TCB 16D5 2.8 0.1 0.69 0.62 0.021 0.028 FolR1 TCB

Example 16 Binding of FolR1 TCB Antibodies to Erythrocytes and T CellActivation in Whole Blood

To prove that there is no spontaneous activation in the absence of FoLR1expressing tumor cells we tested if there is binding of the FolR1 clonesto erythrocytes which might potentially express FolR1. We could notobserve any specific binding of 9D11 IgG, 16D5 IgG and Mov19 IgG toerythrocytes, as negative control DP47 IgG was included (FIG. 8 ).

To exclude any further unspecific binding to blood cells or unspecificactivation via FoLR1 TCB, 9D11 TCB, 16D5 TCB and Mov19 TCB were addedinto whole blood and upregulation of CD25 and CD69 on CD4⁺ T cells andCD8⁺ T cells was analyzed by flow cytometry. DP47 TCB was included asnegative control. No activation of T cells with any of the testedconstructs could be observed by analyzing upregulation of CD25 and CD69on CD4⁺ T cells and CD8⁺ T cells (FIG. 9 ).

Example 17 T-Cell Killing Induced by 36F2 TCB and 16D5 TCB in DifferentMonovalent and Bivalent T-Cell Bispecific Formats

T-cell killing mediated by 36F2 TCB, 16D5 TCB, 16D5 TCB classical, 16D5TCB 1+1 and 16D5 TCB HT antibodies of Hela, Skov-3 (medium FolR1, about70000-90000 copies) and HT-29 (low FolR1, about 10000) human tumor cellswas assessed. DP47 TCB antibody was included as negative control. HumanPBMCs were used as effectors and the killing was detected at 24 h ofincubation with the bispecific antibody. Briefly, target cells wereharvested with Trypsin/EDTA, washed, and plated at density of 25 000cells/well using flat-bottom 96-well plates. Cells were left to adhereovernight. Peripheral blood mononuclear cells (PBMCs) were prepared byHistopaque density centrifugation of enriched lymphocyte preparations(buffy coats) obtained from healthy human donors. Fresh blood wasdiluted with sterile PBS and layered over Histopaque gradient (Sigma,#H8889). After centrifugation (450×g, 30 minutes, room temperature), theplasma above the PBMC-containing interphase was discarded and PBMCstransferred in a new falcon tube subsequently filled with 50 ml of PBS.The mixture was centrifuged (400×g, 10 minutes, room temperature), thesupernatant discarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and stored in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C., 5% CO2 in cell incubator until further use (no longer than 24 h).For the killing assay, the antibody was added at the indicatedconcentrations (range of 0.01 μM-100 nM in triplicates). PBMCs wereadded to target cells at final E:T ratio of 10:1. Target cell killingwas assessed after 24 h of incubation at 37° C., 5% CO2 byquantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct. The results show target-specific killing of allthree FolR1+ target cell lines induced by 36F2 TCB and 16D5 TCB (FIG. 10).

Example 18 Generation of Anti-TIM3 Antibodies

Immunization of mice NMRI mice were immunized genetically, using aplasmid expression vector coding for full-length human Tim-3 byintradermal application of 100 ug vector DNA (plasmid 15304_hTIM3-fl),followed by Electroporation (2 square pulses of 1000 V/cm, duration 0.1ms, interval 0.125 s; followed by 4 square pulses of 287.5 V/cm,duration 10 ms, interval 0.125 s. Mice received either 6 consecutiveimmunizations at days 0, 14, 28, 42, 56, 70, and 84. Blood was taken atdays 36, 78 and 92 and serum prepared, which was used for titerdetermination by ELISA (see below). Animals with highest titers wereselected for boosting at day 96, by intravenous injection of 50 ug ofrecombinant human Tim-3 human Fc chimera, and monoclonal antibodies wereisolated by hybridoma technology, by fusion of splenocytes to myelomacell line 3 days after boost.

Determination of serum titers (ELISA) Human recombinant Tim-3 human Fcchimera was immobilized on a 96-well NUNC Maxisorp plate at 0.3 ug/ml,100 ul/well, in PBS, followed by: blocking of the plate with 2% CroteinC in PBS, 200 ul/well; application of serial dilutions of antisera, induplicates, in 0.5% Crotein C in PBS, 100 ul/well; detection withHRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch/Dianova115-036-071; 1/16 000). For all steps, plates were incubated for 1 h at37° C. Between all steps, plates were washed 3 times with 0.05% Tween 20in PBS. Signal was developed by addition of BM Blue POD Substratesoluble (Roche), 100 ul/well; and stopped by addition of 1 M HCl, 100ul/well. Absorbance was read out at 450 nm, against 690 nm as reference.Titer was defined as dilution of antisera resulting in half-maximalsignal.

Example 19 Characterization Anti-Tim3 Antibodies

ELISA for Tim3 Nunc-Maxi Sorp Streptavidine plates (MicroCoat#11974998/MC1099) were coated by 25 μl/well with Tim3-ECD-His-Biotin(biotinylated with BirA Ligase) and incubated at RT for 1 h whileshaking at 400 rpm rotation. After washing (3×90 μl/well withPBST-buffer) 25 μl aTim3 samples or diluted (1:2 steps) referenceantibody aTim3 F38-2E2 (Biolegend) was added and incubated 1 h at RT.After washing (3×90 μl/well with PBST-buffer) 25 μl/wellsheep-anti-mouse-POD (GE NA9310V) was added in 1:9000 dilution andincubated at RT for 1 h while shaking at 400 rpm rotation. After washing(4×90 μl/well with PBST-buffer) 25 μl/well TMB substrate (Calbiochem,#CL07) was added and incubated until OD 1.5-2.5. Then the reaction wasstopped by addition of 25 μl/well 1N HCL-solution. Measurement tookplace at 370/492 nm. ELISA results are listed as EC50-values [ng/ml] insummary Table 17 below.

Cell ELISA for Tim3 Adherent CHO-K1 cell line stably transfected withplasmid 15312_hTIM3-fl_pUC_Neo coding for full-length human Tim3 andselection with G418 (Neomycin resistance marker on plasmid) were seededat a concentration of 1.2×10E6 cells/ml into 384-well flat bottom platesand grown over night.

At the next day 25 μl/well Tim3 sample or aTim3 reference antibodyF38-2E2 Azide free (Biolegend, 354004) was added and incubated for 2 hat 4 C (to avoid internalization). After washing (3×90 μl/well PBST(BIOTEK Washer: Prog. 29, 1×90) cells were fixed by flicking outresidual buffer and addition of 50 μl/well 0.05% Glutaraldehyde:Dilution 1:500 of 25% Glutaraldehyde (Sigma Cat. No: G5882) in1×PBS-buffer and incubated for 1 h at RT. After washing (3×90 μl/wellPBST (BIOTEK Washer: Prog. 21, 3×90 GreinLysin) 25 μl/well secondaryantibody was added for detection (Sheep-anti-mouse-POD; Horseradish PODlinked F(ab′)₂ Fragment; GE NA9310) followed by 2 h incubation at RTwhile shaking at 400 rpm. After washing (3×90 μl/well PBST (BIOTEKWasher: Prog. 21, 3×90 GreinLysin) 25 μl/well TMB substrate solution(Roche 11835033001) was added and incubated until OD 1.5-2.5. Then thereaction was stopped by addition of 25 μl/well 1N HCL-solution.Measurement took place at 370/492 nm. Cell ELISA results are listed as“EC50 CHO-Tim3”-values [ng/ml] in summary table Table 17 below.

TABLE 17 Binding Affinites of exemplary antibodies ( ELISA and BIACORE)Tim3_ Tim3_ Tim3_ Tim3_ Tim3_ Tim3_ Assay 0018 0021 0028 0026 0033 0038Affinity KD [nM] 3.4/1.1 204/4.1 173/2.8 6.2/1.5 n.f./3.1 7.6/0.6mono/bivalent Tim3 EC50 ELISA [nM] 0.56 0.22 0.501 EC50 ELISA [ng/ml] 9447 37 47 1321 83 EC50 CHO-Tim3 [nM] 0.52 0.32 0.17 EC50 CHO-Tim3 [ng/ml]87 73 53 69 3710 29

Biacore characterization of the Tim3 ABs A surface plasmon resonance(SPR) based assay has been used to determine the kinetic parameters ofthe binding between several murine Tim3 binders as well as commercialhuman Tim3 binding references. Therefore, an anti-mouse IgG wasimmobilized by amine coupling to the surface of a (Biacore) CM5 sensorchip. The samples were then captured and hu/cy Tim3-ECD was bound tothem. The sensor chip surface was regenerated after each analysis cycle.The equilibrium constant K_(D) was finally gained by fitting the data toa 1:1 langmuir interaction model. About 12000 response units (RU) of 30μg/ml anti-mouse IgG (GE Healthcare #BR-1008-38) were coupled onto thespots 1, 2, 4 and 5 of the flow cells 1-4 (spots 1,5 are active andspots 2,4 are reference spots) of a CM5 sensor chip in a Biacore B4000at pH 5.0 by using an amine coupling kit supplied by GE Healthcare.

The sample and running buffer was HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow cell temperature wasset to 25° C. and sample compartment temperature to 12° C. The systemwas primed with running buffer. The samples were injected for 30 secondswith a concentration of 200 μg/ml and bound to the spots 1 and 5 of eachflow cell, allowing the measurement of eight samples in parallel. Then acomplete set of different (monomeric cyno, monomeric human and huFcfused dimeric human Tim3-ECD) concentrations (s. Table X) was injectedover each sample for 240 s followed by a dissociation time of 30/1800 s(s. Table 1). Each analysis cycle (sample capture, spot 1 and 5—Tim3 ECDinjection) was then regenerated with a 30 seconds long injection ofGlycine-HCl pH 1.7. The flow rate was set to 30 μl/min for the wholerun. Finally, the double referenced data was fitted to a 1:1 langmuirinteraction model with the Biacore B4000 Evaluation Software. ResultingK_(D) values are shown in Table 17 and 18.

TABLE 18 Binding affinities determined by Biacore-KD values gained by akinetic SPR measurement.-n.f. means no fit possible, most likely due tono or weak binding. huTim3 K_(D) huTim3Fc K_(D) cyTim3 K_(D) Sample (25°C.) [M] (25° C.) [M] (25° C.) [M] TIM3-0016 3.29E−09 1.09E−09 2.16E−08TIM3-0016 variant 3.40E−09 1.11E−09 4.19E−08 (0018) TIM3-0021 2.04E−074.07E−09 n.f. TIM3-0022 1.26E−07 1.52E−09 2.84E−08 TIM3-0026 6.23E−091.52E−09 n.f. TIM3-0028 1.73E−07 2.77E−09 n.f. TIM3-0030 3.11E−091.28E−09 n.f. TIM3-0033 n.f. 3.05E−09 n.f. TIM3-0038 7.56E−09 5.69E−10n.f. Reference antibody 1.36E−08 7.50E−09 1.68E−07 Biolegend F38-2E2Reference antibody 1.34E−08 7.73E−09 1.41E−07 USB 11E365

Example 20 Generation of Anti-Tim3 Antibody Derivatives

Chimeric antibodies derivatives Chimeric Tim3 antibodies were generatedby amplifying the variable heavy and light chain regions of theanti-TIM3 mouse antibodies Tim3-0016, Tim3-0016 variant (0018),Tim3-0021, Tim3-0022, Tim3-0026, Tim3-0028, Tim3-0030, and Tim3-0033,Tim3-0038 from via PCR and cloning them into heavy chain expressionvectors as fusion proteins with human IgG1 backbones/humanCH1-Hinge-CH2-CH3 with LALA and PG mutations (Leucine 234 to Alanine,Leucine 235 to Alanine, Proline 329 to Glycine) abrogating effectorfunctions and light chain expression vectors as fusion proteins to humanC-kappa. LC and HC Plasmids were then cotransfected into HEK293 andpurified after 7 days from supernatants by standard methods for antibodypurification.

Removal of glycosylation site NYT: Modifying 1 HVR-L1 position inTim3-0016, Tim3_0016 variant (named 0018 or Tim3_0018) by substitutionof N by Q or S Mutations within the variable light vchain region ofTim3_0016 and Tim3_0016 variant (0018) were generated by in vitromutagenesis using Agilent “Quick Change Lightning Site-directedMutagenesis Kit” according manufacturer's instructions. By this methodthe asparagine (N) of the glycoslyation site motif NYT in the lightchain HVR-L1 (SEQ ID NO: 4) was replaced by glutamine (Q) (resulting inSEQ ID NO: 11=Tim3_0016_HVR-L1 variant 1_NQ) or, alternatively, theasparagine (N) was replaced by serine (S) (resulting in SEQ ID NO:12=Tim3_0016_HVR-L1 variant 2_NS). In both glycoslyation site motif NYTwas successfully modified. LC and HC Plasmids coding for the variantswere then cotransfected into HEK293 and purified after 7 days fromsupernatants by standard methods for antibody purification. Thegenerated mutants were tested by ELISA on human Tim3, ELISA oncynomolgus Tim3 and cellular ELISA on adherent CHO-K1 cells expressingfull-length human Tim3. All mutants generated were found to show evenmore functional binding to human TIM3 (human), cyno TIM3 (cyno) or humanTIMR on CHO cells than the parental antibodies Tim3_0016 or theTim3_0016 antibody variant Tim3_0018 respectively.

TABLE 19 Biochem Human Biochem Cyno cellular bindg. CHO-Tim3 EC50[ng/ml] EC50 [ng/ml] EC50 [ng/ml] values values values in relation inrelation in relation to the inflexion to the inflexion to the inflexionsample's point sample's point sample's point Mutants tested max value[ng/ml] max value [ng/ml] max value [ng/ml] aTim3 F38 2E2 73.2 86.3423.0 209878.3 350.2 224.3 aTim3 0018 16.1 15.3 14.6 14.6 26.4 29.4aT3m3 0018MutNQ 12.0 10.8 13.2 10.8 13.4 12.8 aT3m3 0018MutNS 10.3 6.511.9 6.5 11.2 11.1 aTim3 0016 MutNQ 7.6 5.7 8.3 5.7 6.8 5.4 aTim30016MutNS 8.5 6.5 9.7 5.5 9.1 8.5

Example 21 Fluorescent Labeling of Purified Monoclonal Antibody

The fluorescent labeling of the hybridoma derived monoclonal antibodywas carried out by using Alexa Fluor 488 Monoclonal Antibody LabelingKit (manufactured by Invitrogen) according to the manufacturer'sinstructions. After the labeling, each antibody was confirmed to bepositively labeled with Alexa Fluor 488 (hereinafter referred to as“Alexa-488”) by FACSCalibur (manufactured by BD Biosciences) analysisfor TIM-3 expressing RPMI-8226 and Pfeiffer cells.

Example 22 Classification of Binding Epitope Groups Using FACS BasedCompetition Assay

The relation of epitopes between generated anti-TIM3 antibodies and sixanti-TIM3 reference antibodies was analyzed by a FACS based bindingcompetition assay. The TIM3 reference antibodies were the following:antibodies 4177 and 8213 as described in US2012/189617, antibodies1.7E10 and 27.12E12 as described in WO2013/06490; antibody 344823 (Clone344823, manufactured by R&D Systems) and antibody F38-2E2 (CloneF38-2E2, manufactured by BioLegend and R&D Systems). In brief, the testantibody was allowed to interact and bind with the TIM-3 expressingRPMI-8226 cells (ATCC® CCL-155™) and then it was evaluated by flowcytometry method whether another anti-TIM-3 antibody could also bind toTIM-3 expressing cells.

In short human TIM3 expressing RPMI-8226 cells were incubated with BDhuman Fc Block for 10 min at RT and stained in two differentexperimental setups to exclude the impact of the difference in theaffinity of the tested antibodies on the binding:

1) with disclosed purified anti-TIM3 (10 μg/ml in BD staining buffer for0.5 h at 4° C.), which were conjugated with Alexa*488 according to themanufacturer's instructions (Molecular Probes A-20181) with an averageof 2.7 fluorophores per antibody. Then a) unlabeled (1-4) referencerecombinant anti-TIM3 antibodies or Isotype control were added (10μg/ml) for 0.5 h at 4° C. in BD SB and after washing with BD SB stainedwith PE-labeled anti-huFcγ Abs (JIR, 109-116-098, 1:200, 0.5 h at 4° C.in BD SB) or b) PE labeled (5-6) available reference anti-TIM3antibodies or appropriate Isotype controls were added (10 μg/ml) for 0.5h at 4° C. in BD SB. After washing and centrifugation MFI signals ofstained RPMI-8226 cells were analyzed by BD Biosciences FACSCanto flowcytometer.

TABLE 20 Summary of epitope characterzation. Max % inhibition of BindingEpitope Group 1 Epitope Group 3 1a 1b 3a 3b TIM3-0016 TIM3-0018TIM3-0026 TIM3-0022 TIM3-0028 TIM3-0038 clone 4177 1 −9 29  79  −3  0clone 8213 −2   9  9  9  38 29 clone 1-7E10 −5  15 24  0  20  7 clone27-12E12 −1   4 22  40  82 94 clone 344823 0  0  3 102 107 99 cloneF38-2E2 −7  −6  2  77  75 94 100 >90 100 >50 100 >30 100 >20

Results from the FACS based epitope groups mapping show that Tim3_0016and Tim3_0016 variant Tim3_0018 show no binding competition with anytested anti-TIM-3 reference antibodies and it was suggested that theseAbs recognized the new epitope different from the epitopes to which allprevious described TIM3 reference antibodies recognized whereasTim3_0022, Tim3_0026, Tim3_0028 and Tim3_0038 compete to differentextend for binding to surface expressed TIM3 on JRPMI-8226 cells withvarious competitors.

Example 23 Effect of Human Anti-TIM-3 Antibodies on Cytokine Productionin a Mixed Lymphocyte Reaction (MLR)

A mixed lymphocyte reaction was used to demonstrate the effect ofblocking ther TIM-3 pathway to lymphocyte effector cells. T cells in theassay were tested for activation and theier IFN-gamma secretion in thepresence or absence of an anti-TIM-3 mAbs.

Human Lymphocytes were isolated from peripheral blood of healthy donorby density gradient centrifugation using Leukosep (Greiner Bio One, 227288). Briefly, heparinized blood were diluted with the three fold volumeof PBS and 25 ml aliquots of the diluted blood were layered in 50 mlLeukosep tubes. After centrifugation at 800×g for 15 min at roomtemperature (w/o break) the lymphocyte containing fractions wereharvested, washed in PBS and used directly in functional assay orresuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07 cells/mland stored in liquid nitrogen. 1:1 target/responder cell ratio was usedin MLR assay (i.e. each MLR culture contained −2.0E+05 PBMCs from eachdonor in a total volume of 200 μl. Anti-TIM3 monoclonal antibodiesTim3_0016, Tim3_0016 variant (Tim3_0018), Tim3_0021, Tim3_0022,Tim3_0026, Tim3_0028, Tim3_0030, Tim3_0033, Tim3_0038 and F38-2E2(BioLegend), were added to each culture at different antibodyconcentrations. Either no antibody or an isotype control antibody wasused as a negative control and rec hu IL-2 (20 EU/ml) was used aspositive control. The cells were cultured for 6 days at 37° C. After day6 100 μl of medium was taken from each culture for cytokine measurement.The levels of IFN-gamma were measured using OptEIA ELISA kit (BDBiosciences).

The results are shown in Table 21 (IFN-g secretion/release). Theanti-TIM-3 monoclonal antibodies promoted T cell activation andIFN-gamma secretion in concentration dependent manner. The anti-TIM3antibodies Tim3_0021, Tim3_0022, Tim3_0028, and Tim3_0038 reduce releaseof the inflammatory cytokine IFN-gamma) more than the F38-2E2 antibody.Tim3_0016, Tim3_0016 variant (Tim3_0018), Tim3_0033 and Tim3_0038 showeda similar release when compared the F38-2E2 antibody. In contrast,cultures containing the isotype control antibody did not show anincrease in IFN-gamma secretion.

TABLE 21 Percentage anti-Tim3 antibody induced IFNgamma release incomparison to rec hu IL-2 (20 EU/ml) ( = 100%) as positive control andno antibody as negative control (Donors Compound MLR + IL- Isotype F38-Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 Isotype concentration 220U/ml IgG2a 2E2 0016 0018 0021 0022 0026 0028 0030 0033 0038 hlgGl 40μg/ml 2 36 33 36 112 58 25 40 14 35 51 0 10 μg/ml 100 0 26 22 30 108 3816 38  4 30 38 5  1 μg/ml 0  7  7 12 101 18 18 12  3  0  1 0

Example 24 Internalization of Anti-TIM-3 Antibodies into TIM-3Expressing Cells

TIM-3-specific antibodies described herein can be internalized intoTIM-3-expressing cells, including TIM-3 expressing lymphoma, multiplemyeloma and AML cells. For example, the disclosed TIM-3 specificantibodies and fragments thereof are shown to be internalized into recTIM3 CHO cells stabile expressing human TIM-3 as evaluated by cell basedELISA, flow cytometry (FACS) and confocal microscopy.

Stable Tim3-transfected CHO-K1 cells (clone 8) (4×104 cells/well/100 μl)were seeded into 98 well-MTP using fresh culture medium. After overnightcell attachment, cell culture medium was removed and the test antibodieswere added to the cells (10 μg/ml in cell culture medium) and incubatedfor 0.5 hour at 4° C. As reference, a commercial mouse-anti-humanantibody (TIM3 MAB 11E365 (US Biological, T5469-92P) was used. Afterwashing (2× with cell culture medium) and centrifugation cells wereincubated for 3 h at a) 4° C. or b) 37° C. in 200 μl cell culturemedium. Internalization typically occurs at 37° C., but not at 4° C.,which provides another control for the reaction. Then cells were fixatedwith 100 μl/well 0.05% glutharaldehyde (Sigma Cat. No: G5882) in 1×PBSfor 10 min at room temperature (RT). This was followed by three washingsteps with 200 μl PBS-T and secondary antibody sheep-anti-mouse-POD(Horseradish POD linked F(ab′)₂ Fragment; GE NA9310)) were added for 1hour at RT. After the final washing steps (3×PBS-T), TMB substrate wasadded (Roche order no. 11835033001) for 15 min and color development wasstopped using 1N HCl. Final ODs were determined by measurement at450/620 nm in an ELISA reader. This cellular ELISA procedure was usedfor medium throughput evaluation of the internalizing capacity of thetesting antibodies which were purified from hybridoma supernatants.

The percentage of internalization was calculated as follow:Internalization[%]=(1−ODsample_37° C./OD sample_4° C.)*100The results are shown in FIGS. 29A and B for (Internalization). Almostall tested anti-TIM-3 monoclonal antibodies were similar wellinternalized into stable Tim3-transfected CHO-K1 cells after 3 hincubation at 37° C. (not all data shown).

The determination of EC50 internalizing values (time dependency) as wellas comparison of the kinetics of the internalization depending onmono-vs. bivalency was estimated by FACS for selected candidates.

In short, human TIM3 stable expressing CHO-K1 cells were seeded (4×10⁵cells/well/50 μl) into 98 well-v bottom MTP using fresh culture mediumand incubated with Redimune® NF Liquid for 10 min at RT to blockunspecific binding. Then 50 μl/well of selected purified anti-TIM3 (10μg/ml in cell culture medium) were added and incubated for 1 h at 4° C.After washing (with cell culture medium) and centrifugation cells wereincubated for 0.25, 0.5, 1, 2, 3, 4, 6 and 24 h at a) 4° C. or b) 37° C.in 200 μl cell culture medium. Than cells were washed with PBS/l % BSAand secondary antibody Alexa Fluor 488 Goat-anti-mouseIgG, F(ab)₂ wereadded for 1 hour at 4° C. After washing and centrifugation 125 μl ofCellFix (BD Bioscience, 1:1000) were added and MFI signals of stainedcells were analyzed by BD Biosciences FACSCanto flow cytometer.

The percentage of internalization was calculated as follow:Internalization[%]=(1−MFIsample_37° C./MFIsample_4° C.)*100

Example for the evaluation of time dependent internalization ofanti-TIM3 antibodies Tim3_0016, Tim3_0016 variant (Tim3_0018),Tim3_0021, Tim3_0028, Tim3_0030, Tim3_0033, Tim3_0038 on RPMI-8226 cells(ATCC® CCL-155™): The presently disclosed anti-TIM3 antibodies areinternalized rapidly into TIM3 expressing RPMI-8226 cells (ATCC®CCL-155™) at a high level. The experiments were conducted as describedabove with TIM3 expressing RPMI-8226 cells (ATCC® CCL-155™) instead ofrec CHOK1 cells expressing huTIM-3. Results are shown in the Table 22.The following antibodies were used as TIM3 reference antibodies:antibody 8213 as described in US2012/189617, antibody 27.12E12 asdescribed in WO2013/06490. Tim3_0016, Tim3_0016 variant (Tim3_0018),Tim3_0038 were used as human IgG1 chimeric versions.

TABLE 22 Percentage internalization at the indicated time point (0 minset as 0 percent). Percentage internalization of anti-TIM3 antibodiesAntibody 30 Min 60 Min 120 Min 240 Min 26 h 8213 22 22 43 52 72 27.12E1219 22 25 46 59 Tim3_0016 33 52 55 66 87 Tim3_0018 39 41 80 70 88Tim3_0021 70 75 74 78 77 Tim3_0028 50 59 67 68 83 Tim3_0033 75 81 82 8280 Tim3_0038 22 20 45 46 63

The results show that the tested antibodies are rapidly internalized athigh percentage compared to reference antibodies on RPMI-8226 cells(ATCC® CCL-155™).

Example 25 Binding of Anti-TIM-3 Antibodies to Isolated Human MonocytesExpressing TIM-3

CD14+ Monocytes were isolated from anticoagulated peripheral blood ofhealthy donors by density gradient centrifugation using Ficoll-Paque (GEHealthcare) (see General Protocols in the User Manuals or visitwww.miltenyibiotec.com/protocols) and subsequent positive selection viaCD14 MicroBeads. First the CD14+ cells are magnetically labeled withCD14 MicroBeads. Then the cell suspension is loaded onto a MACS® Columnwhich is placed in the magnetic field of a MACS Separator. Themagnetically labeled CD14+ cells are retained in the column. Theunlabeled cells run through, this cell fraction is depleted of CD14+cells. After removal of the column from the magnetic field, themagnetically retained CD14+ cells can be eluted as the positivelyselected cell fraction. After centrifugation at 200×g for 10 min at roomtemperature the monocytes were harvested and used directly in bindingassay or resuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07cells/ml and stored in liquid nitrogen.

As shown in the literature Monocytes express constitutively TIM3 ontheir surface. 1×10⁵ CD14+ isolated human monocytes (50 μl/well) wereput into 98 well-v bottom MTP in fresh culture medium and incubated withRedimune® NF Liquid for 15 min at RT to block unspecific binding. Than50 μl/well of disclosed anti-TIM3 mAbs or reference anti-TIM-3 mAbs344823 (R&D) and F38-2E2 (BioLegend) (10 μg/ml in cell culture medium)were added and incubated for 1 h at 4° C. Than cells were washed withPBS/l % BSA and secondary antibody PE-labeled Goat-anti-mouse F(ab′)2were (Jackson Lab 115-006-072) added for 1 hour at 4° C. After washingand centrifugation MFI signals of stained cells were analyzed by BDBiosciences FACSCanto flow cytometer.

The specific binding was calculated as follow:Specific Binding [MFI]=Geom. Mean MFIsample−Geom. Mean MFIisotypecontrol

The results are shown in Table 8: (Binding to human Monocytes). TIM3clones Tim3_0016, Tim3_0018, Tim3_0020, Tim3_0028 and Tim3_0038 bind tohuman monocytes of different donors even better than the referenceanti-TIM-3 Abs.

TABLE 23 Binding to human Monocytes. donor1 donor2 donor3 (CD14+)(CD14+) (CD14+) Tim3 0016 2122 1634 1690 Tim3 0018 2326 1818 1943 Tim30020 1917 1377 1462 Tim3 0021 1134 951 1197 Tim3 0022 1468 1111 1235Tim3 0026 1665 1016 900 Tim3 0030 1411 419 466 Tim3 0038 1637 1368 1401Tim3 0028 1351 950 1607 Tim3 0033 480 328 595 M-IgG2b 0 13 0 M-IgG1 14455 213 <TIM-3>PE Mab, M-IgG1 (Cl 516 493 460 F38-2E2; Biolegend)<TIM-3>PE Mab, Rat IgG2A 1010 917 814 (Clone 344823, R&D) Rat-IgG2A-PE71 68 70

Example 26 Binding of Anti-TIM-3 Antibodies to Isolated Cyno MonocytesExpressing TIM-3

CD14+ Monocytes were isolated from cynomolgus monkey anticoagulatedperipheral blood (Covance) by density gradient centrifugation usingFicoll-Paque (GE Healthcare) (see General Protocols in the User Manualsor visit www.miltenyibiotec.com/protocols) and subsequent positiveselection via NHP CD14 MicroBeads. First the CD14+ cells aremagnetically labeled with CD14 MicroBeads. Then the cell suspension isloaded onto a MACS® Column which is placed in the magnetic field of aMACS Separator. The magnetically labeled CD14+ cells are retained in thecolumn. The unlabeled cells run through, this cell fraction is depletedof CD14+ cells. After removal of the column from the magnetic field, themagnetically retained CD14+ cells can be eluted as the positivelyselected cell fraction. After centrifugation at 200×g for 10 min at roomtemperature the monocytes were harvested and used directly in bindingassay or resuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07cells/ml and stored in liquid nitrogen.

As shown in the literature Monocytes express constitutively TIM3 ontheir surface. 1×10⁵ CD14+ isolated cyno monocytes (50 μl/well) were putinto 98 well-v bottom MTP in fresh culture medium and incubated withRedimune® NF Liquid for 15 min at RT to block unspecific binding. Than50 μl/well of Alexa488 labeled anti-TIM3 (10 μg/ml in cell culturemedium) were added and incubated for 1 h at 4° C. After washing andcentrifugation MFI signals of stained cells were analyzed by BDBiosciences FACSCanto flow cytometer.

The specific binding was calculated as follow:Specific Binding [MFI]=Geom. Mean MFIsample−Geom. Mean MFIisotypecontrol

The results are shown in Table 9 (Binding to Cyno Monocytes). TIM3clones Tim3_0016, Tim3_0018, Tim3_0026, Tim3_0028 and, Tim3_0030 bind tocyno monocytes of different cyno donors.

TABLE 24 Binding to Cyno Monocytes. cyno1 cyno2 cyno3 (16719M) (17435M)(30085F) CD14+ CD14+ CD14+ AF + PI 75 83 84 HumTIM-3 Alexa488 R&D 158121 143 (34482) Rat-IgG2A-Alexa488 84 86 91 hum TIM-3 A488 F38-2E2 135136 124 (NOVUS Biol) M-IgG1-Alexa 488 72 82 83 Tim3 _0016-A488 157 177187 Tim3 _0016 variant 0018-A488 301 480 417 Tim3 0022-A488 115 134 138Tim3 0026-A488 137 184 197 Tim3 0028-A488 3936 2996 4090 Tim3 0038-A48897 107 120 Tim3_0020-A488 274 378 354 Tim3 0021 A488 348 473 399 Tim30030 A488 119 163 144 Tim3 0033 A488 71 81 83 TIM-3 (4177) A488 78 83 85TIM-3 (8213) A488 75 83 87

Example 27 Binding of Anti-TIM-3 Antibodies to NHL and MM Cell LinesExpressing TIM-3

The binding capacity of disclosed anti-TIM3 antibodies and two anti-TIM3reference antibodies clones (1) 4177 and (2) 8213 (Kyowa) was analyzedby a FACS. In short human TIM3 expressing B cell lymphoma cells(exemplified as Pfeiffer cells) and multiple myeloma cells (exemplifiedas RPMI-8226 cells) were incubated with BD human Fc Block for 10 min atRT to block unspecific binding. Then 2×10⁵ cells (50 μl/well) were putinto 98 well-v bottom MTP and 50 μl/well of Alexa488 labeled anti-TIM3(10 μg/ml in BD Staining buffer) were added and incubated for 1 h at 4°C. After washing and centrifugation MFI signals of stained cells wereanalyzed by BD Biosciences FACSCanto flow cytometer.

The specific binding was calculated as follow:Specific Binding [MFI]=Geom. Mean MFIsample−Geom. Mean MFIisotypecontrolThe results are shown in FIGS. 2A and 2B (Binding to RPMI-8226 andPfeiffer cells).

Example 10: Cytotoxic Activity of Anti-TIM-3 Antibodies on TIM-3Expressing NHL and MM Cells

TIM3-specific antibodies conjugated with Pseudomonas exotoxin (PE 24)effectively kill TIM3-expressing cells. The cytotoxic activity ofdisclosed anti-TIM3 antibodies and one commercial available anti-TIM3reference antibody clone 11E365 (available from US Biological) wasanalysed with Promega CellTiter-Glo Luminescent Cell Viability Assay. Inshort to 5×103 (50 μl/well in 98 well MTP, in triplicate) recombinantCHO K1 stabile expressing human TIM-3 or 2 x104 cells (50 μl/well in 98well MTP, in triplicate) human TIM3 expressing B cell lymphoma cells(exemplified as Pfeiffer cells) or multiple myeloma cells (exemplifiedas RPMI-8226 cells) were added 25 μl/well 1:5 serial dilution ofdisclosed anti-TIM-3 antibodies with the highest concentration of 10μg/ml or appropriate media to untreated cells or Isotype control tountargeted treated cells. Treatment ranges from 10 μg/ml to 1 ng/ml intriplicate. All antibodies were used as full length mouse Fcγ versions.For conjugation of the conjugation of the Pseudomonas exotoxin 10 μg/mlof mouse Fcγ fragment specific Fabs conjugated with PE 24 were added andincubated for 3 days at 37° C. Cycloheximide as a known proteinsynthesis inhibitor in eukaryotes was used as positive control.Viability of treated cells were measured with Promega CellTiter-GloLuminescent Cell Viability Assay.

The cytotoxic activity was calculated as follow:Rel. Inhibition [%]=(1−(Esampel−E negative control)/(E positivecontrol−E negative control))*100

The results are shown in Table 25.

TABLE 25 Cytotoxic activity of anti-TIM3 mAbs on TIM-3 expressingrecombinant, NHL and MM cell lines in sandwich format. Antibodies andreferences (all anti TIM3 antibodies conjugated IC50 [nM] to adeimunized Pseudomonas recTIM-3 Pfeiffer RPMI- exotoxin A) CHO cellscells 8226 Tim3_0016 0.04 0.09 0.55 Tim3_0016 variant 1 0.05 0.10 0.66(Tim3_0018) Tim3_0020 0.07 0.11 >64 Tim3_0021 0.04 0.10 5.9 Tim3_00220.02 0.07 0.36 Tim3_0023 0.03 0.08 >64 Tim3_0026 0.03 0.08 >64 Tim3_00300.03 0.10 >64 Tim3_0033 0.11 0.20 0.79 Tim3_0038 0.01 <0.002 0.16 USBiol. Clone 11E365 0.7 1.2 1.1 Cells w/o Ab — — — Cells + <mFc> Fab PE —— — IgG2A + <mFc> Fab PE — — — Cycloheximide 135 181 245

All tested TIM3 clones are highly potent (IC50 range 0.01-0.2 nM) onrecombinant CHO K1 stabile expressing human TIM-3 and Pfeiffer cellsexpressing high and moderate levels of TIM-3 and even more potent intheir cytotoxic activity than the strong internalizing referenceanti-TIM-3 Ab clone 11E365, US Biological. TIM3 clones 0016, 0018, 0021,0022, 0033 and 0038 are also potent on RPMI-8226 cells expressing 5 foldlower TIM-3 level compare to recombinant CHO TIM-3 cells.

Example 28: Comparison of the Cytotoxic Activity of Disclosed Anti-TIM3Antibodies Vs. Two Anti-TIM3 Reference Antibodies 1.7.E10 and 27-12E12(as Described in WO2013/06490)

The cytotoxic activity of disclosed anti-TIM3 antibodies and twoanti-TIM3 reference antibodies the TIM3 reference antibodies 1.7E10 and27.12E12 as described in WO2013/06490 was analysed with PromegaCellTiter-Glo Luminescent Cell Viability Assay as described above. Allantibodies were used as full length human IgG1 format including thehuman Fcgamma part. In this experiment conjugation of the Pseudomonasexotoxin was achieved via human Fcγ fragment specific Fabs conjugatedwith PE 24 (10 μg/ml) which were added and incubated for 5 days at 37°C.

The results are shown in Table 26.

TABLE 26 Comparison of cytotoxic activity of anti-TIM3 mAbs on TIM-3expressing NHL and MM cell lines. Antibodies and references Pfeiffercells RPMI-8226 cells (all anti TIM3 antibodies Rel. Rel. conjugated toa deimunized Max. IC50 Max. IC50 Pseudomonas exotoxin A) killing [nM]killing [nM] Cycloheximide  100 [%] 271  100 [%] 111 1.7E10 60.3 [%]0.68 65.7 [%] 2.544 27-12E12 75.7 [%] 0.02 86.6 [%] 0.111 Tim3_0016 84.9[%] 0.05 86.6 [%] 0.063 Tim3_0016 variant 82.9 [%] 0.06 88.1 [%] 0.081(Tim3_0018) Tim3_0026 78.3 [%] <0.02 83.1 [%] 0.067 Tim3_002 82.6 [%]<0.02 83.8 [%] 0.047 Isotype Control hIgG1  3.2 [%] N.A  0.4 [%] N.A

All disclosed TIM3 clones are highly active (IC50 range 0.02-0.08 nM) onPfeiffer and RPMI-8226 cells expressing TIM-3 and even more potent intheir cytotoxic activity than the strong internalizing referenceanti-TIM-3 Ab clone 27-12E12. All antibodies were compared asPseudomonas exotoxin (PE24) conjugates using the same Pseudomonasexotoxin under the same conditions.

Example 28 Cytotoxic Activity of Fab-PE24 Constructs of DisclosedAnti-TIM3 Antibodies on MM, NHL and AML Cell Lines (Expressing TIM3, butnot PSMA)

The cytotoxic activity was analysed with Promega CellTiter-GloLuminescent Cell Viability Assay as described above. 1:5 serialdilutions of Fab-fragments of disclosed anti-TIM3 antibodies directlyconjugated to PE24 with the highest concentration of 50 μg/ml orappropriate media to untreated cells or non-binding anti-PSMA Fab-PE24control to untargeted treated cells were incubated with 7.5×103 Pfeiffercells or 2×10³ RPMI-8226 cells (50 μl/well in 98 well MTP) for 4 days at37° C. Treatment ranges from 50 μg/ml to 8 ng/ml in triplicate.Cycloheximide was used as positive control.

The results are shown in Table 27.

TABLE 27 Cytotoxic activity of Fab-PE24 constructs of disclosed anti-TIM3 antibodies on MM, NHL and AML cell lines. RPMI-8226 Karpas-299 CMKTF-1 MOLM-13 Antibodies Max. IC50 Max. IC50 Max. IC50 Max. IC50 Max.IC50 and references killing [nM] killing [nM] killing [nM] killing [nM]killing [nM] (all anti TIM3 antibodies conjugated to a deimunizedPseudomonas exotoxin A) Cycloheximide  100 [%] 281  100 [%] 113  100 [%]149.0  100 [%] 207  100 [%] 156 Anti_PSMA 10.5 [%] N.A. 40.1 [%] N.A.8.98 [%] N.A. 5.27 [%] N.A. 18.9 [%] N.A. Tim3_0022 99.1 [%] 1.9 98.8[%]  10 67.1 [%] 255 58.6 [%] 299 58.5 [%] 579 Tim3_0016 99.3 [%] 1.199.2 [%]  4 64.8 [%] 225 54.2 [%] 534 62.7 [%] 459

All tested Fab-PE24 constructs of disclosed anti-TIM3 antibodies arehighly potent (IC50 range 1-10 nM) on MM (RPMI-8226) and NHL(Karpas-299) cells expressing moderate level of TIM-3 and demonstratesignificant cytotoxic activity on AML cell lines (CMK, TF-1, MOLM-13)expressing very low levels of TIM-3.

Example 29 Cytotoxic Activity of Immuno Conjugates (Pseudomonas ExotoxinA Conjugates (Fab-PE24 Constructs) of Disclosed Anti-TIM3 on PrimaryLeukemic Stem/Progenitor AML Cells from Relapsed/Refractory Patients

CD34+ cells from peripheral blood of relapsed/refractory patients wereobtained from AllCells, LLC, Alameda, Calif. After confirmation ofpurity and viability of all samples (purity range 84-94% and viabilityrange 95-99%) the expression level of TIM-3 was evaluated by FACS asdescribed in Example 7 using anti-TIM-3 mAbs 344823 (R&D). (see FIG. 31). All tested (4/4) primary leukemic stem/progenitor (CD34+) AML samplesfrom relapsed/refractory patients demonstrate homogeneous expression ofTIM-3 at different levels.

For the evaluation of cytotoxic activity of Fab-PE24 constructs ofdisclosed anti-TIM3 clones 0016 and 0022 on primary CD34+ AML cells1×10⁴ cells (50 μl/well in 98 well MTP, in triplicate) were incubatedwith 1:5 serial dilutions of Fab-fragments with the highestconcentration of 50 μg/ml or appropriate media to untreated cells ornon-binding anti-PSMA Fab-PE24 control to untargeted treated cells for 3days at 37° C. Cycloheximide was used as positive control. Cytotoxicactivity was analysed with Promega CellTiter-Glo Luminescent CellViability Assay as described above in Example 28.

The results are shown in Table 28. (Cytotoxic activity of Fab-PE24constructs of disclosed anti-TIM3 antibodies on primary CD34+ AMLcells).

TABLE 28 Cytotoxic activity of Fab-PE24 constructs of disclosedanti-TIM3 antibodies on primary CD34 + AML cells). Dl; AML D2; AML D3;AML D4; AML CD34 + PB0136 CD34 + PB0142 CD34 + PB0135 CD34 + PB0193cells cells cells cells Antibodies Max. IC50 Max. IC50 Max. IC50 Max.IC50 and references killing [nM] killing [nM] killing [nM] killing [nM](all anti TIM3 antibodies conjugated to a deimunized Pseudomonasexotoxin A) Cycloheximide 100 [%] 212 100 [%] 262 100 [%] 121 100 [%]208 anti-PSMA  2 [%] N.A.  8 [%] N.A.  18 [%] N.A.  12 [%] N.A. TIM-30022-cFP  38 [%] >691   75 [%] 107  31 [%] >691   57 [%] 375 TIM-30016-cFP  48 [%] >691   79 [%]  30  44 [%] >691   69 [%] 116

Fab-PE24 constructs of anti-TIM3 antibodies Tim3_0016 and Tim3_0022 arehighly potent on (2/4) primary AML samples (PB0142 and PB0135) (IC50range 30-116 nM) and demonstrate significant cytotoxic activity on all(4/4) primary leukemic stem/progenitor (CD34+) AML cells expressingdifferent levels of TIM-3.

Example 30 Comparison of Potency of Fab-PE24 Constructs of SelectedAnti-TIM3 Antibodies on NHL and MM Cell Lines

The evaluation of cytotoxic activity of sortase coupled Fab-PE24constructs of selected disclosed anti-TIM3 antibodies was analysed withPromega CellTiter-Glo Luminescent Cell Viability Assay as describedabove in Example 28.

The results are shown in Table 29.

TABLE 29 Cytotoxic activity of Fab-PE24 constructs of selected anti-TIM3antibodies on NHL and MM cells. Antibodies and references (all anti TIM3antibodies conjugated to a deimunized Pfeiffer cells RPMI-8226 cellsPseudomonas Max. IC50 Max. IC50 exotoxin A) killing [nM] killing [nM]Cycloheximide  100 [%] 271.1  100 [%] 153 anti-PSMA 25.2 [%] N.A. 21.5[%] N.A. TIM-3 0022 99.9 [%] 1.58 99.6 [%] 2.14 TIM-3 0016 99.6 [%] 0.7799.2 [%] 0.61 TIM-3 0021 98.4 [%] 2.15 99.1 [%] 3.61 TIM-3 0033 99.8 [%]5.30 99.7 [%] 5.73 TIM-3 0038 99.6 [%] 0.47 98.3 [%] 0.32

High cytotoxic potency was demonstrated with Fab-PE24 constructs of allselected disclosed anti-TIM3 antibodies (IC50 range 0.3-5 nM) on NHL(Pfeiffer) and MM (RPMI-8226) cells expressing moderate level of TIM-3.

The highest cytotoxic activity was observed with Fab-PE24 constructs ofdisclosed anti-TIM3 antibodies Tim3_0016 and Tim3_0038.

Example 31 Comparison of Cytotoxic Activity of Fab-PE24 Construct Vs.Total-IgG-Amatoxin Conjugate of the Same Clone of Disclosed Anti-TIM-3Antibody on Pfeiffer Cells

The evaluation of cytotoxic activity of conjugated Fab-PE24 construct ofdisclosed anti-TIM3 clone 0016 vs. total IgG of the same cloneconjugated with Amatoxin (according to the procedures described inWO2012/041504 (conjugated via the 6′C-atom of amatoxin amino acid 4,particularly via an oxygen atom bound to the 6′C-atom of amatoxin aminoacid, and wherein the TIM3 antibody is connected by a linker via a ureamoiety) was analysed with Promega CellTiter-Glo Luminescent CellViability Assay as described above in Example 12. The results are shownin Table 30.

TABLE 30 Cytotoxic activity of Fab-PE24 construct vs. total IgG-Amatoxinconjugate of anti-TIM3 clone 0016 on NHL cells Pfeiffer cells Max.killing IC50 [nM] Cycloheximide  100 [%] 163 Isotype hIgG1 Amatoxin   28[%] N.A. TIM-3 0016- Amatoxin 93.3 [%] 0.81 TIM-3 0016-PE24 99.8 [%]0.25

Example 32 Patients and Tumor Sample Processing

Freshly excised solid tumor lesions and malignant effusions werecollected from 34 patients with non-small cell lung cancer, 7 patientswith ovarian cancer and 1 patient with renal cell carcinoma (RCC)between. The solid tumor lesions were dissociated mechanically anddigested using accutase (PAA), collagenase IV (Worthington),hyaluronidase (Sigma), and DNAse type IV (Sigma) directly afterexcision. Single-cell suspensions were prepared. The cellular fractionof malignant effusions was isolated by density gradient centrifugationusing Histopaque-1119 (Sigma). All samples were stored in liquidnitrogen until further usage. The study was approved by the localEthical Review Board (Ethikkommission Nordwestschweiz).

Example 33 Tumor Sample Characterization

All tumor samples were comprehensively characterized by multicolor flowcytometry.

The following antibodies were used for flow cytometric analysis:α-CD4-PE, α-CD8-PE-Cy7, α-CD11b-PerCP-eFluor710, α-CD45-PE-Cy7,α-CD45-PerCP-Cy5.5, α-CD137-FITC, α-BTLA-Biotin, α-CTLA-4-PE,α-ICOS-FITC, α-IFN-γ-FITC, α-Lag-3-APC (all eBioscience), α-CD3-PECF594,α-CD25-BV605, α-CD69-FTC, α-Epcam-FITC, α-granzyme B-PE, α-activecaspase 3-PE, α-PD-1-BV605, Steptavidin-BV711 (all BD Bioscience),α-CD45RA-BV421, α-CCR7-AlexaFluor647, α-FoxP3-AlexaFluor647,α-Tim-3-BV421, α-Tim-3-BV605 (all Biolegend). Dead cells were stainedwith LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit or LIVE/DEAD®Fixable Blue Dead Cell Stain Kit (Invitrogen). For intracellularstainings Fixation and Permabilization Buffers from eBioscience wereused. Samples were acquired for flow cytometric analysis on a BD LSRFortessa. The human IL-2, IFN-γ and TNF ELISA sets were all obtainedfrom BD Bioscience.

CD8⁺ and CD4⁺ T cells (CD45+CD3+) were characterized for the expressionof the surface markers PD-1, Tim-3, CTLA-4, Lag-3, BTLA, CD25, CD69,CD137, ICOS, CD45RA and CCR7. Tumor cells (CD45-Epcam⁺) werecharacterized for the expression of FolR1 comparing the binding of aFolR1 specific antibody with its matched isotype control. Only samplesthat were positive for FolR1 expression were used for treatment withFolR1-TCB, and samples expressing EpCAM for treatment with catumaxomab,respectively.

Example 34 Ex Vivo Treatment of Tumor Samples with FolR1-TCB

FolR1 positive tumor digests or malignant effusions were thawed, washedand plated in 96-well flat bottom cell culture plates (BD Falcon) with adensity of 3×10 cells/200 μl/well in complete medium (DMEM+SodiumPyruvate (1 mM)+MEM non essential AA (1×)+L-Glutamin (2mM)+Penicillin/Streptomycin (100 ng/ml)+2-Mercaptoethanol (50nM)+Ciproxin (1 mg/ml)+10% human Serum). The samples were cultured inthe presence or absence of FolR1-TCB or DP47 TCB at a concentration of 2nM for 24 h. Activation of CD8⁺ and CD4⁺ T cells (CD45⁺CD3⁺) uponFolR1-TCB treatment was determined by multicolor flow cytometry bymeasuring the expression of the cell surface markers CD25, CD69, CD137,ICOS, PD-1 and Tim-3. Furthermore the expression of granzyme B and IFN-γwas determined by intracellular staining. The concentration of IL-2 inthe cell culture supernatants was measured by ELISA (human IL-2 ELISAset, BD OptEIA) following the instructions of the manufacturer.

Example 35 Ex Vivo Treatment of Tumor Samples with Catumaxomab

The trifunctional TCB catumaxomab (Removab®) was obtained fromFresenius. The experimental conditions were similar as indicated abovefor FolR1-TCB. Briefly, EpCAM positive tumor digests or malignanteffusions were cultured in the presence or absence of catumaxomab at aconcentration of 10 ng/ml for 24 h. Analysis of CD8⁺ and CD4⁺ positive Tcells (CD45+CD3+) was performed as described above.

Example 36 Killing Assay

To determine the FolR1-TCB induced tumor cell killing, 3×10⁴CFSE-labelled Skov3 cells were cocultured with tumor samples in thepresence or absence of FolR1-TCB at a concentration of 2 nM for 24 h in96-well flat bottom cell culture plates. The E:T ratio (E: effectorCD45+CD3+ cells; T: target FolR1⁺ cells from tumor and added Skov3cells) was adjusted to 1:1 in each well and the cell number of the addedtumor samples was calculated for each sample according to priorcharacterization by flow cytometry. Cell death of Skov3 cells wasdetermined by flow cytometry by measuring activated caspase 3 and thelive/dead marker Live/Dead-near-IR. The assay was performed intriplicates. The FolR1-TCB mediated killing was calculated according tothe following equation: % of specific killing=100−[(% of Skov3 livecells in FolR1-TCB treated sample/% of Skov3 live cells in untreatedsample)×100].

To compare the FolR1-TCB-induced killing capacity of T-cells betweentumor samples, and to exclude additional factors suppressing T-cellfunctionality, such as expression of PD-L1 on the tumor cells, weexogenously added CFSE-labeled FolR1⁺ Skov3 cells to the tumor digestsand adjusted the E:T ratio to 1:1, essentially as described above. Wethen measured the FolR1-TCB-induced killing of CFSE-labeled Skov3 cells,which allowed us to also include FolR1⁻ tumor samples into the analysis.As some tumors from the initial cohort could not be used to characterizeTCB-mediated tumor cell killing due to a very low amount of effectorcells, a separate cohort of 12 tumor digests and 5 malignant effusionsfrom 15 non-small cell lung cancer (NSCLC) and two epithelial ovariancarcinoma (EOC) patients was analyzed. All samples were characterizedfor their CD3⁺ effector and FolR1⁺ target cell content (FIG. 39 ). Tumorcell killing of CD3⁺ T-cells from patients was compared withPBMC-derived T-cells from healthy donors. A substantial heterogeneity intumor cell killing between individual patients was observed (26±11.8%)after 24 h (FIG. 12O). Of note, CD3⁺ T-cells from healthy donors induceda significantly better killing than TILs (42.8±9.7%, p=0.013). Exposureto a control TCB with no binding to a tumor antigen (DP47-TCB) did notinduce any tumor cell killing.

Example 37 Polyclonal Stimulation with Anti-CD3/CD28 Antibodies

A 96-well flat-bottom plate was precoated with 0.5 μg/ml anti-CD3ε(clone OKT3, Biolegend) for 2 hrs at 37° C. Afterwards, the antibodysolution was removed and the plate washed extensively. Frozen tumorsuspensions were thawed, washed and cultured at 3×10⁵ cells/200 μl/wellin complete medium with 2 μg/ml anti-CD28 antibody (clone 28.2,eBioscience) for 24 hrs. After 24 hrs of incubation cells werecollected, washed and analyzed by flow cytometry for expression ofactivation markers e.g. CD25 and T cell effector functions e.g. granzymeB and IFN-γ on CD8⁺ T cells. Supernatants were collected for IL-2, IFN-γand TNF-α ELISA which was performed according to the manufacturer'sinstructions.

Example 38 Restoring of T Cell Function by PD-1 Blockade

Tumor digests were stimulated by agonistic anti-CD3 and anti-CD28antibodies as described above in the presence or absence of 10 μg/mlanti-PD-1 antibody (MDX5C4) per well and incubated for 24 hrs. After 24hrs cells were collected, washed and analyzed by flow cytometry.Supernatants were collected for IL-2, IFN-γ and TNF-α ELISA which wasperformed according to the manufacturer's instructions.

Example 39 Activation of T Cells in Tumor Digests and MalignantEffusions by FolR1 TCB

The T cell bispecific antibodies engaging CD3 and folate receptor 1(Mov19 based FolR1-TCB and the control antibody DP47-TCB were providedby Roche Glycart. The anti-PD-1 antibody 5C4 is described in U.S. Pat.No. 8,008,449. The anti-Tim3 antibody F38-2EL was used. For flowcytometric characterization of FolR1 expression the antibodyanti-FolR1-APC (aa25-233) from LifeSpanBiosciences and its matchedisotype control (Biolegend) were used. Tumor lesions from 15 patientswith FolR1⁺ tumors were characterized for T cell activation induced byFolR1 TCB. The samples consisted of 9 single cell suspensions and 6malignant effusions derived from patients with NSCLC (n=7), ovariancancer (n=7), and renal cell cancer (n=1). The amount of CD3⁺ T cellsand of FolR1⁺ tumor cells was highly variable between patients (CD3+:mean 33.9% t standard deviation of 16.6%, FolR1⁺: 17.1% t 16.8%).Characterization of the expression of the inhibitory receptors PD-1,Tim-3, CTLA-4, Lag- and BTLA on T cells revealed a large heterogeneityamong patients (FIG. 11A-B). While the tumor-infiltrating CD8⁺ T cellsshowed high levels of PD-1, Tim-3 and CTLA-4 (31.6%±25%; 22.2%±20.8% and18.7%±14.4%, respectively), Lag-3 and BTLA were only expressed on aminority of cells in all patients of this cohort (3.5%±4.9% and2.3%±1.7%, respectively). Inhibitory receptors on CD4⁺ T cells weredistributed similarly, with a slightly more prominent expression ofCTLA-4.

To determine FolR1-TCB induced T cell activation, tumor samples werecultured in the presence or absence of FolR1-TCB or the control TCBDP-47. Then, T cells were characterized by multicolor flow cytometry forexpression of activation markers and T cell effector functions, asdescribed above. FIG. 12A-O reveals a large heterogeneity in FolR1-TCBinduced T cell activation between patients. In particular, while thevast majority of patients expressed CD69 already at baseline,upregulation of CD25, CD137, and ICOS, varying from 9-80%, 2.5-50% and3.5-71%, respectively was observed. Acquisition of effector functionssuch as IFN-γ secretion, CD107 degranulation and expression of granzymeB was observed, ranging from 3.7-59%, a fold change of 1-7 or 1.3-64,respectively (FIG. 12A-I). The inhibitory receptors PD-1 and Tim-3 werefurther upregulated as a marker of activation upon FolR1-TCB treatment,irrespective of their baseline expression. Exposure to TCB DP-47 did notinduce any T cell activation. The upregulation of CD25 and ICOS inducedby FolR1-TCB stimulation was significantly stronger in peripheral CD8⁺T-cells from healthy donors than for tumor-derived CD8⁺ cells (p=0.002and p<0.001, respectively; FIG. 12J, FIG. 12L, FIG. 12M). The secretionof T-cell effector cytokines IFN-γ, IL-2, and TNF upon FolR1-TCBstimulation was largely diminished amongst TILs in the majority oftumors compared with PBMCs from healthy donors (p=0.0047, p<0.001, andp=0.006, respectively; FIG. 12N). FolR1-TCB-induced perforin secretionwas highly variable in TILs, and severely impaired in a subset ofpatients (FIG. 12N).

Similarly, despite a lower upregulation of granzyme B, FolR1-TCB inducedactivation and acquisition of effector functions of CD4⁺ T cells (FIG.25A-I). To assess whether the abundance of intra-tumoral T cells orFolR1 expression impacts on T cell activation upon TCB exposure, theupregulation of activation markers was correlated to the E:T ratio (E:effector CD45⁺CD3⁺ T cells; T: FolR1⁺ cells) and to the percentage andto the level of tumor antigen expression of FolR1⁺ cells (FIG. 13A-C).The latter was determined by the mean fluorescence intensity of FolR1 ontumor cells (CD45⁻EpCAM⁺) using flow cytometry (FIG. 13C). However,neither of these parameters did influence T cell activation, i.e., evenlow amounts of FolR1⁺ cells, high E:T ratios, or poor T-cellinfiltration have been sufficient for an efficient upregulation ofactivation and functional markers. In addition, the presence ofpotentially immune-suppressive cell populations such as regulatoryT-cells or immature myeloid cells did not influence T-cell activation orT-cell function.

Example 40 FolR1-TCB Induced T Cell Activation Inversely Correlates withExpression of PD-1 and Tim-3

High expression of inhibitory receptors has been described as a hallmarkof exhausted T cells. Therefore, a dysfunctional state oftumor-infiltrating T cells may impact efficacy of the FolR1 TCB and maybe responsible, at least in part, for heterogeneous T cell activationupon TCB exposure. To this end, the co-expression of inhibitoryreceptors, as determined at baseline, was correlated to FolR1 TCBinduced upregulation of activation markers and T cell effectorfunctions. Both PD-1 and Tim-3 expression on CD8⁺ T cells therebynegatively correlated with T cell activation determined by expression ofCD25, CD137 and ICOS. CD8⁺ T cells with a high expression of PD-1 orTim-3 showed a marginal effect upon FolR1-TCB treatment, while T cellswith a low expression of these inhibitory receptors could be stronglyactivated upon treatment with FolR1-TCB (FIG. 14A-I). Measurement ofFolR1-TCB induced IL-2 secretion normalized to the content of T cells inthe samples revealed the same dependencies on PD-1 and Tim-3 expression(FIG. 15A-C), while FolR1-TCB induced upregulation of granzyme B wasless dependent on prior expression of these inhibitory receptors (FIG.14J-L). Interestingly, the baseline expression of CTLA-4, Lag-3 and BTLAon CD8⁺ T cells did not correlate with FolR1-TCB induced T cellactivation (FIG. 26A-C). Expression of inhibitory receptors on CD4⁺ Tcells was much less predictive for FolR1-TCB induced CD4⁺ T cellactivation compared to the expression of the same receptors on CD8⁺ Tcells.

Example 41 FolR1-TCB Induced Tumor Cell Killing Inversely Correlateswith Expression of PD-1 and Tim-3

To investigate FolR1-TCB induced killing of tumor cells at an adjustedE:T ratio of 1:1, CFSE-labelled Skov3 cells were exogenously added tothe tumor digests which contain a previously determined amount of CD3⁺ Tcells using multicolor flow cytometry. FolR1-TCB induced killing ofSkov3 cells was determined by measuring activated caspase 3 and alive/dead marker. In line with the FolR1-TCB induced T cell activationas measured by CD25 up-regulation, the specific killing upon FolR1-TCBexposure negatively correlated with single or co-expression of PD-1 andTim-3 on CD8⁺ T cells. Furthermore, FolR1-TCB induced killing was alsoinfluenced by the baseline expression of CTLA-4 and the co-expression ofPD-1 and CTLA-4. However, the impact of CTLA-4 expression on FolR1-TCBinduced tumor cell killing was less pronounced compared to PD-1 andTim-3 expression.

Example 42 Treatment of Fresh Tumor Lesions with Catumaxomab—Activationof Tumor-Infiltrating T Cells Using Catumaxomab and Correlation withExpression of Inhibitory Receptors

To determine to which extent catumaxomab induces T cell activation andto confirm the findings described above using a second, independent Tcell bispecific molecule, 4 tumor digests from patients with NSCLC wereexposed to catumaxomab, a trifunctional bispecific antibody recognizingCD3 on T cells and EpCAM on tumor cells. Then, T cells werecharacterized by flow cytometry for expression of activation markers andT cell effector functions (FIG. 17A-D). Validating our data above forFolR1-TCB, we observed a striking heterogeneity in catumaxomab induced Tcell activation. Accordingly, the baseline expression of inhibitoryreceptors differed between the patients (FIG. 17E-H).

Analysis of T cell activation and effector function upon treatment withcatumaxomab revealed two groups of patients according to PD-1 and/orTim-3 expression on CD8⁺ T cells confirming our findings with FolR1-TCB(FIG. 18A-R). PD-1^(low), Tim-3^(low), and, even more pronounced, bothPD-1^(low)/Tim-3^(low) expressing cells, failed to be activated bycatumaxomab, whereas PD-1_(high), Tim-3^(high), andPD-1^(high)/Tim-3^(high) T cells substantially upregulated CD25, CD69,CD137, ICOS, granzyme B and IFN-γ.

Example 43 Polyclonal Stimulation of Tumor-Infiltrating T Cells byCD3/CD28—Immune Phenotyping of Tumor-Infiltrating T Cell Subsets inNon-Small Cell Lung Cancer Samples

We investigated the expression of co-inhibitory T cell receptors anddifferentiation markers on tumor-infiltrating CD3+CD8⁺ and CD3+CD4⁺ Tcell subsets from 34 patients NSCLC using multicolor flow cytometry. Themajority of tumors showed a high expression of the inhibitory receptorPD-1 (FIG. 19A-B), a major regulator of T cell exhaustion. Of note,expression of other checkpoint inhibitors such as Tim-3, CTLA-4, LAG-3or BTLA showed substantial variation between T cells obtained fromdifferent tumors (FIG. 19A-B).

Example 44 Cumulative Expression of Inhibitory Receptors Defines T CellDysfunction

In this Example, polyclonal stimulation was used in a sub-optimal doseto assess the impact of inhibitory receptors on T cell function. Theeffect of stimulation with agonistic anti-CD3 and anti-CD28 antibodieson T cell activation, as exemplified by CD25 expression, and on T celleffector function as analyzed by IFN-γ, TNF-α and IL-2 production aswell as granzyme B expression varied substantially between patients asdetermined by flow cytometry (FIG. 20A-B) and ELISA (FIG. 20C-E). Ofnote, we observed different levels of T cell function, varying from Tcell populations that exhibit a largely preserved T cell function (i.e.,sustained CD25 and granzyme B expression, as well as IL-2, IFN-γ andTNF-α production) to those with abrogated T cell function (loss of CD25and granzyme B expression and of cytokine production).

To analyze the impact of multiple inhibitory receptors on T cellfunctionality we defined the inhibitory receptor (iR) score as a markerfor the cumulative expression of inhibitory receptors on T cells. Tothis end, the percentage of expression of PD-1, Tim-3, CTLA-4, Lag-3 andBTLA was analysed in all NSCLC samples and a score based on the medianand interquartile ranges of each expressed receptor was defined andcalculated for each sample (e.g., FIG. 21F). Tumor-infiltrating CD8⁺ Tcells expressing a high iR score indicating expression of multipleinhibitory receptors showed a marginal effect upon polyclonalstimulation, correlating with their highly dysfunctional state, whereasT cells with a low iR score could be strongly activated upon polyclonalstimulation (FIG. 21A-E). Upregulation of T cell effector functions,indicated by IL-2, IFN-γ and TNF-α production, not only correlated withthe cumulative expression of inhibitory receptors but similarly withPD-1 and Tim-3 expression as well with the co-expression of bothreceptors (FIG. 22A-I), indicating a significant contribution of PD-1and Tim-3 to T cell dysfunction.

Example 45 Inhibitory Receptor Expression

Single and cumulative expression of inhibitory receptors increases withtumor progression. The expression of inhibitory receptors correlatedwith tumor stage and tumor progression. The number of PD-1, Tim-3 andLAG-3 positive cells was clearly increased in advanced tumor stages(FIG. 21G-K). No clear correlation was observed for the expression ofCTLA-4, which may indicate that this receptor acts via a differentinhibitory mechanism. BTLA was generally expressed at a low level andonly a small increase was found in advanced tumor stages (FIG. 21K). Asignificant increase in the cumulative expression of inhibitoryreceptors, as reflected by the iR score, was observed in patients withnodal positive cancers and advanced tumor stages whereas primary tumorsize did not significantly correlate with the iR score (FIG. 21L-M).These data suggest a gradual and continuous upregulation of inhibitoryreceptors, during tumor progression, which are most likely involved in Tcell exhaustion in NSCLC.

Inhibitory receptors are gradually expressed on tumor-infiltrating Tcells. To explore the role of simultaneous expression of distinctinhibitory receptors on single T cells, the concomitant expression ofthese receptors in CD8⁺ T cells (FIGS. 32, 33 ) relative to theexpression of any of the five analyzed receptors was analyzed.Expression is shown as heat map, displaying the percentage of expressionfor the individual patients (FIG. 32 ) or as a radar plot, which showsthe expression as mean and standard deviation of the four respectivereceptors on CD8⁺ T cells, pregated for the fifth, indicated immunecheckpoint (FIG. 33 ). CD8⁺PD-1⁺ T cells on average expressed the lowestpercentages of other inhibitory receptors, whereas CD8⁺BTLA⁺ T cellsexpressed all of the four other inhibitory receptors at high levels,indicating that BTLA marks a particularly exhausted T cell subset (FIGS.32, 33 ). An increase in the number of co-expressed inhibitory receptorswas observed from CD8⁺Tim-3⁺ T cells over CD8⁺CTLA-4⁺ T cells toCD8⁺LAG-3⁺ T cells (FIGS. 32, 33 ). These findings suggest a gradualacquisition of inhibitory receptors with PD-1 as a broadly expressed,early marker, while BTLA is upregulated rather late during T cellexhaustion.

Example 46 Blockade of PD-1 can Partially Restore T Cell Function

Rescue of T cell function by PD-1 blocking antibodies depends on thelevel of PD-1 expression. As we found a clear correlation between theexpression of inhibitory receptors, particularly PD-1 and Tim-3, and Tcell activation upon polyclonal stimulation, blockade of the PD-1 orPD-1/Tim-3 pathways might restore T cell function. However, addition ofa blocking antibody to PD-1 (5C4) or combined blockade of PD-1 and Tim-3upon stimulation with agonistic anti-CD3 and anti-CD28 antibodies couldrestore T cell effector function such as production and secretion ofIL-2, IFN-γ and TNF-α only in some patients whereas in other patientsonly a marginal effect was seen (FIG. 23A-D). As observed in a chronicmurine LCMV infection model (Blackburn et al., PNAS 105(39):15016(2008)), we identified PD-1^(hi) and PD-1^(int) subsets intumor-infiltrating CD8⁺ T cells from NSCLC patients. In brief,PD-1^(hi), PD-1^(int), and PD-1^(neg) subsets could be identified basedon their measured fluorescence intensity. Cells from 33 patients wereanalysed for PD-1 expression to define uniform parameters forreproducible discernment of the three subsets. The analysis covered thewhole spectrum of PD-1 expression levels and included tumor samples withclearly distinguished PD-1^(neg) or PD-1^(hi) populations. This allowedto set the gates for this analysis, which was then applied to allsamples.

Only PD-1^(int) expressing T cell subsets appeared to be rescued inactivation upon PD-1 or combined PD-1/Tim-3 blockade, while no effect inT cell activation was observed upon blockade in PD-1^(hi) cells (FIG. 24). The latter may exhibit a more exhausted phenotype which appears to beresistant to PD-1 blockade alone.

This finding was confirmed in T cells activated by FolR1 TCB. T cellswere stimulated with FolR1 as described above. Blockade of PD-1 furtherstrengthened FolR1-TCB induced T cell activation of T cells from asubset of patients.

Measurement of FolR1-TCB induced IFN-γ, TNF and IL-2 secretionnormalized to the content of T cells in the samples revealed that inpatient cell populations with a substantial amount of PD-1^(hi)expressing (approximately >15%) cells were not able to secrete thesecytokines. In contrast, cytokine secretion could be induced in mostpatient cell populations with a lower amount of PD-1^(hi) expressing(approximately <15%) cells (FIG. 27A-C). In the latter group, additionof a blocking antibody to PD-1 or combined blockade of PD-1 and Tim-3upon stimulation by FolR1-TCB stimulation increased production of IL-2,IFN-γ and TNF-α (FIG. 28A-F). The PD-1^(hi) expressing subset thereforemay exhibit a more exhausted phenotype which appears to be resistant toPD-1 blockade alone.

Thus, T cell effector functions such as production of IL-2, IFN-γ andTNF-α could be restored in TILs from some NSCLC patients, whereas inother patients only a marginal recovery of T cell functions could beachieved. The increase in cytokine production upon exposure toanti-CD3/CD28 stimulation in combination with the PD-1 blocking antibodywas compared to the percentage of PD-1^(hi) CD8⁺ T cells from the PD-1positive population per patient. The increase in cytokine expressionupon PD-1 blockade inversely correlated with the percentage of PD-1^(hi)T cells, indicating that patients expressing larger numbers of PD-1^(hi)T cells respond poorly to PD-1 blockade alone (FIG. 24A-C). As T celldysfunction correlates with the expression of multiple inhibitoryreceptors (i.e., patients with a high iR score) and response to a PD-1directed therapy correlates with the expression levels of PD-1 on CD8⁺ Tcells, we further analyzed the expression of Tim-3, CTLA-4, LAG-3 andBTLA in PD-1^(hi) and PD-1^(int) CD8⁺ T cells. Remarkably, PD-1^(hi) Tcells expressed significantly higher levels of additional receptorscompared to PD-1^(int) subsets (FIG. 34 ). Thus, PD-1^(hi) andPD-1^(int) may identify two distinct T cell populations where PD-1^(hi)T cells may exhibit a more exhausted phenotype, which cannot berecovered by PD-1 blockade alone.

The data presented herein for the first time provides a comprehensivephenotypical and functional analysis of tumor-infiltrating CD8⁺ T cellsfrom patients with NSCLC. The data shows that these cells mainly possessan effector memory phenotype (CCR7-CD45RAlow) and show largeheterogeneity in expression of inhibitory receptors such as PD-1, Tim-3,CTLA-4, LAG-3 and BTLA. Nevertheless, a clear increase in the number ofreceptors expressed on tumor-infiltrating lymphocytes (TILs) from latestage tumors was observed, which reflects the progress of T celldysfunction during tumor development. The data presented herein showsthat the effector functions of TILs were impaired in the vast majorityof patients, and that impairment correlated with the expression ofinhibitory receptors. To recover T cell function in a clinicallyrelevant setting we combined polyclonal T cell stimulation withantibody-mediated inhibition of PD-1. The effect of PD-1 blockade on Tcell functionality varied between TILs from different patients, butcould be predicted by assessing the percentage of CD8⁺ T cellsexpressing PD-1 at high levels. Here, we could demonstrate that thefunctionality of TILs can be correlated with and is largely affected bythe number and expression level of inhibitory receptors. Of note, even Tcells expressing low levels of inhibitory receptors showed some degreeof impaired functionality, as the secretion of IL-2 was impaired in thevast majority of patients. Overall the activation and effector functionof CD8⁺ T cells inversely correlated with the cumulative expression ofinhibitory receptors, indicating a direct contribution of differentinhibitory pathways to T cell dysfunction in NSCLC.

Our analysis of five inhibitory receptors on tumor infiltrating CD8⁺ Tcells showed a clear increase of the single and cumulative expression ofthese inhibitory receptors in tumor tissues from NSCLC patientspresenting with tumor-positive lymph nodes and advanced tumor stages.Expression of CTLA-4 differed from the other four receptors with thehighest percentage of positive cells at early stages, which may indicatea distinct role of CTLA-4 in regulating T cell immunity (Topalian etal., Safety, activity, and immune correlates of anti-PD-1 antibody incancer. N. Engl. J. Med. 366, 2443 (Jun. 28, 2012)). Co-expressionanalysis of additional inhibitory receptors on single cells, relative tothe expression of one given receptor, showed a gradual expression, withearly and late upregulation of PD-1 and BTLA, respectively. This mayreflect the dynamic process of T cell exhaustion.

The findings presented herein underscore the clinical relevance ofinhibitory receptor expression during NSCLC tumor progression,associated with progressive failure of immune control of tumor growth.We document here two populations of CD8⁺ tumor-infiltrating T cellscharacterized by different levels of PD-1 expression (PD-1^(hi) andPD-lint subsets). The occurrence of PD-1^(hi) T cells did not correlatewith the percentage of PD-1 expression. Interestingly, we observed thatthe effect of PD-1 blockade correlated with the levels of PD-1expression, with minimal effects on responsiveness of TILs with highproportions of PD-1hi subpopulations. These findings are in line withexperiments in a murine, chronic LCMV infection model where the subsetof PD-1^(int) DbGP33-specific CD8⁺ T cells could be restored upon PD-1blockade. In contrast, the PD-1^(hi) subset appeared more “exhausted,”i.e., exhibited signs of functional exhaustion, and responded poorly toPD-1 blockade. Thus, the level of PD-1 expression may represent a novelmarker to define distinct T cell subsets in human cancers and, may serveas a predictor of responses to treatment with PD-1 blocking antibodies.

Example 47 Activation of T-Cells from Healthy Donors and Cancer Patientsby FolR1-TCB

To assess the effect of FolR1-TCB on T-cell activation peripheral bloodmononuclear cells (PBMCs) from healthy donors were co-cultured with theFolR1⁺ ovarian cancer cell line Skov3 (FIG. 40A). Upon exposure toincreasing concentrations of FolR1-TCB ranging from 0.6 pM to 2 nM for24 h we observed a strong activation of CD8⁺ T-cells with upregulationof CD25, CD137, and ICOS. In addition, T-cells secreted IL-2, IFN-γ, andTNF. Exposure to DP47-TCB, a TCB directed against an irrelevant antigen,did not induce any T-cell activation (FIGS. 40B and C).

Example 48 Inhibitory Receptor Expression is Highly Diverse inTumor-Infiltrating CD8⁺ T-Cells

As tumor-resident T-cells frequently display a highly dysfunctionalphenotype, the observed heterogeneity in T-cell activation amongdifferent patients after FolR1-TCB stimulation may be due to an impairedTIL functionality. A hallmark of dysfunctional T-cells in both chronicviral infections and in tumors is the overexpression of inhibitoryreceptors. To this end, we determined the expression of the immunecheckpoints PD-1, Tim-3, CTLA-4, Lag-3, and BTLA on tumor-infiltratingCD8⁺ T-cells in all tumor samples. We observed a high diversity infrequency and combined expression of these receptors amongst differenttumors; PD-1 was found to be the most prominent inhibitory receptor withthe highest percentage of expression (60.2±30%), followed by Tim-3(29.5±24.4%), CTLA-4 (24.6±17.6%), Lag-3 (7.0±5.9%), and BTLA (3.9±2.6%)(FIG. 35F). As described previously in a murine chronic viral infectionmodel (Blackburn et al., Proc Natl Acad Sci USA 2008; 105(39):15016-21)and, as shown herein, in human tumors, the PD-1⁺ population could bedivided into a PD-1^(hi) and a PD-1^(int) expressing subpopulation (FIG.35A). Analysis of additional inhibitory receptors expressed on theseparticular subsets showed a significantly higher expression of all otherinhibitory receptors, including Tim-3, CTLA-4, Lag-3, and BTLA, in thePD-1^(hi) subpopulation as compared with the expression of thesereceptors in the PD-1^(int) and PD-1^(neg) subsets (FIG. 36A-D).Therefore, we used the percentage of PD-1^(hi) T-cells in the CD8⁺subset as a surrogate marker for the cumulative expression of inhibitoryreceptors. The tumor samples were divided according to the frequency ofPD-1^(hi) cells into two groups with high (PD-1^(hi) abundant tumors)and low frequencies of PD-1^(hi) expressing T-cells (PD-1^(hi) scarcetumors), respectively. A cut-off value of 30% PD-1^(hi) expression waschosen to separate the two groups. The percentage of PD-1^(hi) cellsranged from 39.1-60.5% in the PD-1^(hi) abundant (49.5±7.9%) and from2.65-19.5% in the PD-1^(hi) scarce group (8.4±5.7%; FIG. 36E). Thecut-off value was validated in a second cohort of 14 NSCLC and 2 ovariancancer patients with a similar distribution in the frequency ofPD-1^(hi) cells, where we observed comparable results upon polyclonalstimulation by anti-CD3/anti-CD28 antibodies (FIG. 39 ).

Example 49 FolR1-TCB-Induced T-Cell Activation Largely Depends on theLevel of PD-1 Expression on CD8⁺ T-Cells

We analyzed whether the expression of inhibitory receptors could becorrelated with a diminished T-cell functionality upon FolR1-TCBtreatment. Consistent with the results described in Example 41 above,FolR1-TCB-induced T-cell activation, as exemplified by CD25, CD137, andICOS expression (p=0.028; p<0.001, and p=0.008, respectively), andT-cell effector functions, indicated by IFN-γ, IL-2, TNF, as well asperforin secretion, were significantly impaired in PD-1^(hi) abundanttumors compared with PD-1 scarce tumors (p=0.019; p=0.007; p=0.028, andp=0.029, respectively; FIG. 37A-G). Similarly, PD-1^(hi) abundant tumorsdisplayed a significantly reduced cytotoxicity upon FolR1-TCBstimulation whereas a strong tumor cell killing could be observed in themajority of PD-1^(hi) scarce tumors (p=0.021; FIG. 37H).

Example 50 PD-1 Blockade Restores FolR1-TCB-Induced T-Cell Function Onlyin PD-1^(hi) Scarce Tumors

As the level of PD-1 expression on TILs correlates with the efficacy ofFolR1-TCB, we analyzed whether blockade of the PD-1/PD-L1 axis incombination with FolR1-TCB treatment might be able to restore T-cellfunction. We found that upon combined treatment with FolR1-TCB and thePD-1 blocking antibody nivolumab (MDX5C4) secretion of the effectorcytokines IFN-γ, TNF, and IL-2 as well as perforin could be increasedonly in some of the PD-hi scarce tumors. In contrast, in PD-1^(hi)abundant tumors PD-1 blockade failed to elicit any response (FIG.38A-D). Of note, cytotoxic tumor cell killing could neither be improvedin T-cells from PD-1^(hi) scarce nor from PD-1^(hi) abundant tumors bythe additional PD-1 blockade (FIG. 38E).

The examples set forth herein describe the immuno-modulatory capacity ofa CD3×FolR1-specific TCB in primary cancer lesions from patients withnon-small cell lung cancer (NSCLC), epithelial ovarian carcinoma (EOC)and renal cell carcinoma (RCC). Compared with fully functionalperipheral T-cells from healthy donors, we observed a substantialheterogeneity in FolR1-TCB-induced tumor cell killing and T-cellactivation among different human tumor samples, resulting in partial orcomplete impairment of T-cell function in the majority of patients.Comprehensive analysis of inhibitory receptor expression on the cellsurface of intratumoral T-cells revealed that the efficacy of T-cellactivation by FolR1-TCB inversely correlated with the expression levelsof PD-1. Patients with PD-1^(hi) abundant tumors displayed impairedT-cell activation and effector function upon FolR1-TCB treatment.Additionally, these patients did not respond to PD-1 blockade incontrast to their PD-1^(hi) scarce expressing counterparts. Thus, thebioactivity of bispecific antibodies is considerably hampered by T-celldysfunction, which is orchestrated, at least in part, by the sustainedand highly diverse expression of inhibitory receptors.

We observed a strong upregulation of T-cell activation markers, effectorcytokine secretion and tumor cell killing upon FolR1-TCB stimulation inPBMCs from healthy donors (FIG. 40 ). In stark contrast, however, T-celleffector functions largely varied and were generally diminished inintratumoral T-cells. Particularly, killing capacity and effectorcytokine production was significantly lower in TILs with complete lossof IL-2 production and severely impaired TNF and IFN-γ secretion in themajority of tumors. We documented the expression of the inhibitoryreceptors PD-1, Tim-3, CTLA-4, Lag-3, and BTLA on intratumoral CD8⁺T-cells. PD-1 displayed the broadest expression of all analyzedinhibitory receptors. Observations from chronic murine LCMV infectionsby Blackburn suggest the presence of functionally distinct PD-1 positiveT-cell subsets, which can be separated on the basis of MFI levels, usingflow cytometry (Blackburn et al., PNAS 105(39):15016 (2008)). Of note,PD-1^(hi) T-cell subsets displayed a high co-expression of Tim-3 andCTLA-4 and to a lesser extent of Lag-3 and BTLA, while their PD-1^(int)counterparts expressed only low levels of other inhibitory receptors,comparable to PD-1^(neg) T-cells. The frequency of PD-1^(hi) CD8⁺T-cells differed largely between patients and allowed us to discriminatebetween PD-1^(hi) abundant and scarce tumors. In contrast to patientswith a PD-1^(hi) scarce phenotype, FolR1-TCB-mediated T-cell activationand tumor cell killing was significantly impaired in tumors displaying aPD-1^(hi) abundant phenotype. These data extend and confirm previousobservations that the activation and effector function of CD8⁺ T-cellscorrelates with the co-expression of multiple immune checkpoints(Sakuishi et al., J Exp Med 2010; 207(10):2187-94; Fourcade et al., JExp Med 2010; 207(10):2175-86; Grosso et al., J Immunol 2009;182(11):6659-69; Matsuzaki et al., Proc Natl Acad Sci USA 2010;107(17):7875-80; Fourcade et al., Cancer Res 2012; 72(4):887-96). Thefrequency of PD-1^(hi) T-cells may therefore be useful as a surrogatemarker for the functionality of TILs upon TCB activation as well asserve as a predictive marker for the therapeutic responses to TCBtreatment. This immune profile could guide the selection of patients whoare likely to respond to immunotherapy such as TCBs. Its correlationwith clinical benefits remains to be determined in prospective clinicalinterventions.

A promising avenue to improve the therapeutic efficacy of TCBs lies inthe blockade of inhibitory signals on T-cells. As PD-1 was the mostprominently expressed inhibitory receptor in all tumors analyzed weassessed whether PD-1 blockade could enhance T-cell effector functionsupon TCB activation. Of note, we observed increased secretion ofeffector cytokines upon combined FolR1-TCB and anti-PD-1 treatment,though only in PD-1^(hi) scarce tumors. Thus, novel therapeuticstrategies, exploring the transformation of PD-1^(hi) intoPD-1^(int)T-cells to increase the susceptibility to PD-1/PD-L1 blockade,are clearly needed.

Remarkably, we observed no improvement on tumor cell killing uponconcomitant PD-1 blockade in all of the tumor samples. Thus, blockade ofa single immune checkpoint may not be sufficient to restore thecytolytic capacity of TILs. In a mouse tumor model, however, blockade ofthe PD-1/PD-L1 axis has been shown to increase T-cell infiltration intotumors (Curran et al., Proc Natl Acad Sci USA 2010; 107(9):4275-80), acharacteristic of this treatment, which could not be addressed by our invitro approach. Thus, the therapeutic effect of PD-1 blockade in vivomight not only result from improving T-cell cytotoxicity of residualintratumoral T-cells, but from the sustained functionality of newlyinfiltrating T-cells. TCB-induced T-cell activation has been shown toupregulate PD-1 expression, which may lead to secondary resistance inthe presence of PD-L1 expressed on both tumor cells and infiltratingimmune cells as recently demonstrated both with a Her2-specific TCB andwith a carcinoembryonic antigen-(CEA) specific TCB (Junttila et al.,Cancer Res 2014; 74(19):5561-71; Osada et al., Cancer Immunol Immunother2015). Importantly, blockade of the PD-1/PD-L1 axis could completelyrestore TCB-induced T-cell function both in vitro and in a mouse tumormodel. These observations indicate that co-administration of checkpointinhibitors is capable of preventing secondary resistance, which may addto the dysfunctional state of TILs and limit the therapeutic efficacy ofTCBs. Further work is clearly needed to determine optimal combinationregimens of checkpoint inhibitors and TCBs. It will also be crucial toidentify inhibitory and activating T-cell-receptors with non-redundantfunctions as potential therapeutic targets.

Our findings clearly indicate that bispecific antibodies such asFolR1-TCB are capable of causing T-cells to upregulate co-stimulatorymolecules, produce inflammatory cytokines, and acquire cytolyticfunction. We have observed different states of T-cell dysfunction, whichare orchestrated, at least in part, by the expression of inhibitoryreceptors and, in some instances, reduce the effectiveness of the TCB.As FolR1-TCB-induced effector functions could only be partially restoredby PD-1 blockade, our results suggest a rather complex immuneregulation, which utilizes multiple and eventually non-redundantpathways to maintain T-cell dysfunction within the tumor environment.

SEQUENCES

Amino Acid Sequences of Exemplary Embodiments

1) FolR binders useful in common light chain format, variable heavychain

Description Sequence Seq ID No 16A3QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  1WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYYAGVTPFDYWGQGTLVTVSS 18D3QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  2WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYYTGGSSAFDYWGQGTLVTVS 15H7QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  3WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYYLFSTSFDYWGQGTLVTVSS 15B6QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  4WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYYIGIVPFDYWGQGTLVTVSS 21D1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  5WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYYVGVSPFDYWGQGTLVTVSS 16F12QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  6WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNFTVLRVPFDYWGQGTLVTVSS 15A1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE  7WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNYYIGVVTFDYWGQGTLVTVSS 15A1_CDR1 SYYMH  8 15A1_CDR2IINPSGGSTSYAQKFQG  9 15A1_CDR3 NYYIGVVTFDY 10 19E5QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 11WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEWRRYTSFDYWGQGTLVTVSS 19E5_CDR1 SYYMH  8 19E5_CDR2IINPSGGSTSYAQKFQG  9 19E5_CDR3 GEWRRYTSFDY 12 19A4QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 13WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGWIRWEHFDYWGQGTLVTVSS 19A4_CDR1 SYYMH  8 19A4_CDR2IINPSGGSTSYAQKFQG  9 19A4_CDR3 GGWIRWEHFDY 14 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 15WVGRIKSKIDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSS 16D5_CDR1 NAWMS 16 16D5_CDR2RIKSKIDGGITDYAAPVKG 17 16D5_CDR3 PWEWSWYDY 18 15E12EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 19WVGRIKSKIDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSYFDYWGQGTLVTVSS 15E12_CDR1 NAWMS 16 15E12_CDR2RIKSKIDGGITDYAAPVKG 17 15E12_CDR3 PWEWSYFDY 20 21A5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 21WVGRIKSKIDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWAWFDYWGQGTLVTVSS 21A5_CDR1 NAWMS 16 21A5_CDR2RIKSKIDGGITDYAAPVKG 17 21A5_CDR3 PWEWAWFDY 22 21G8EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE 23WVGRIKSKIDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWAYFDYWGQGTLVTVSS 21G8_CDR1 NAWMS 16 21G8_CDR2RIKSKIDGGITDYAAPVKG 17 21G8_CDR3 PWEWAYFDY 24 19H3QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 25WMGIINPSGGSTSYAQKFQGRVIMIRDTSTSTVYMELSSLRSEDTAVYYCARTGWSRWGYMDYWGQGTLVTVSS 19H3_CDR1 SYYMH  8 19H3_CDR2IINPSGGSTSYAQKFQG  9 19H3_CDR3 TGWSRWGYMDY 26 20G6QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 27WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEWIRYYHFDYWGQGTLVTVSS 20G6_CDR1 SYYMH  8 20G6_CDR2IINPSGGSTSYAQKFQG  9 20G6_CDR3 GEWIRYYHFDY 28 20H7QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 29WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVGWYRWGYMDYWGQGTLVTVSS 20H7_CDR1 SYYMH  8 20H7_CDR2IINPSGGSTSYAQKFQG  9 20H7_CDR3 VGWYRWGYMDY 30

2) CD3 binder common light chain (CLC)

Description Sequence Seq ID No common QAVVTQEPSLTVSPGGTVTLTCGSSTGA 31CD3 light VTTSNYANWVQEKPGQAFRGLIGGTNKR chain (VL)APGTPARFSGSLLGGKAALTLSGAQPED EAEYYCALWYSNLWVFGGGTKLTVL commonGSSTGAVTTSNYAN 32 CD3 light chain_CDR1 common GTNKRAP 33 CD3 lightchain_CDR2 common ALWYSNLWV 34 CD3 light chain_CDR3 commonQAVVTQEPSLTVSPGGTVTLTCGSSTGA 35 CD3 light VTTSNYANWVQEKPGQAFRGLIGGTNKRchain APGTPARFSGSLLGGKAALTLSGAQPED (VLCL) EAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLIS DFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS

3) CD3 binder, heavy chain

Seq ID Description Sequence No CD3 EVQLLESGGGLVQPGGSLRLSCAAS 36 variableGFTFSTYAMNWVRQAPGKGLEWVSR heavy IRSKYNNYATYYADSVKGRFTISRD chain (VH)DSKNTLYLQMNSLRAEDTAVYYCVR HGNFGNSYVSWFAYWGQGTLVTVSS CD3 heavy TYAMN 37chain (VH)_CDR1 CD3 heavy RIRSKYNNYATYYADSVKG 38 chain (VH)_CDR2CD3 heavy HGNFGNSYVSWFAY 39 chain (VH)_CDR3 CD3 fullEVQLLESGGGLVQPGGSLRLSCAAS 40 heavy GFTFSTYAMNWVRQAPGKGLEWVSR chainIRSKYNNYATYYADSVKGRFTISRD (VHCH1)_ DSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSC CD3 ASTKGPSVFPLAPSSKSTSGGTAAL 84 constantGCLVKDYFPEPVTVSWNSGALTSGV heavy HTFPAVLQSSGLYSLSSVVTVPSSS chain CH1LGTQTYICNVNHKPSNTKVDKKVEP KSC

4) FolR binders useful for crossfab Format

Seq ID Description Sequence No 11F8_VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE 41WMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAVFYRAWYSFDYWGQGTTVTVSS 11F8_VH_CDR1 SYAIS 42 11F8_VH_CDR2GIIPIFGTANYAQKFQG 43 11F8_VH_CDR3 AVFYRAWYSFDY 44 11F8_VLDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKL 45LIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYT SPPPTFGQGTKVEIK11F8_VL_CDR1 RASQSISSWLA 46 11F8_VL_CDR2 DASSLES 47 11F8_VL_CDR3QQYTSPPPT 48 36F2_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 49WMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSS 36F2_VH_CDR1 SYYMH  8 36F2_VH_CDR2IINPSGGSTSYAQKFQG  9 36F2_VH_CDR3 SFFTGFHLDY 50 36F2_VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 51LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY TNEHYYTFGQGTKVEIK36F2_VL_CDR1 RASQSVSSSYLA 52 36F2_VL_CDR2 GASSRAT 53 36F2_VL_CDR3QQYTNEHYYT 54 9D11_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 55WMGIINPSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYCARGDFAWLDYWGQGTLVTVSS9D11_VH_CDR1 SYYMH  8 9D11_VH_CDR2 IINPSGGPTSYAQKFQG 56 9D11_VH_CDR3GDFAWLDY 57 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 58QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRTFGQGTKVEIK9D11_VL_CDR1 RSSQSLLHSNGYNYLD 59 9D11_VL_CDR2 LGSNRAS 60 9D11_VL_CDR3MQASIMNRT 61 9D11_VL DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 62N95S QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMSRTFGQGTKVEIK9D11_VL MQASIMSRT 63 N95S_CDR3 9D11_VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 64 N95QQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMQRTFGQGTKVEIK9D11_VL MQASIMQRT 65 N95Q_CDR3 9D11_VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 66 T97AQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRAFGQGTKVEIK9D11_VL MQASIMNRA 67 T97A 9D11_VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG 68 T97NQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CMQASIMNRNFGQGTKVEIK9D11_VL MQASIMNRN 69 T97N_CDR3 5D9_VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 70WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYCARSYIDMDYWGQGTLVTVSS5D9_VH_CDR1 SYYMH  8 5D9_VH_CDR2 IINPSGGSTSYAQKFQG  9 5D9_VH_CDR3SYIDMDY 71 5D9_VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 72LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQD NWSPTFGQGTKVEIK5D9_VL_CDR1 RASQSVSSSYLA 52 5D9_VL_CDR2 GASSRAT 53 5D9_VL_CDR3 QQDNWSPT73 6B6_VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLE 74WMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTA VYYCARSYVDMDYWGQGTLVTVSS6B6_VH_CDR1 SYYMH  8 6B6_VH_CDR2 IINPSGGSTSYAQKFQG  9 6B6_VH_CDR3SYVDMDY 75 6B6_VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 76LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQD IWSPTFGQGTKVEIK6B6_VL_CDR1 RASQSVSSSYLA 52 6B6_VL_CDR2 GASSRAT 53 6B6_VL_CDR3 QQDIWSPT77 14E4_VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 78WVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSYVEWYAFDYWGQGTLVTVSS 14E4_VH_CDR1 SYAMS 79 14E4_VH_CDR2AISGSGGSTYYADSVKG 80 14E4_VH_CDR3 DSSYVEWYAFDY 81 14E4_VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR 82LLIYGASSRATGIPDRFSGSGSGTDSTLTISRLEPEDFAVYYCQQP TSSPITFG QGTKVEIK14E4_VL_CDR1 RASQSVSSSYLA 52 14E4_VL_CDR2 GASSRAT 53 14E4_VL_CDR3QQPISSPIT 83

5) CD3 binder useful in crossfab Format

Seq ID Description Sequence No CD3 heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWV 36 chain(VH)SRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS CD3 heavy  TYAMN 37 chain (VH)_CDR1CD3 heavy RIRSKYNNYATYYADSVKG 38 chain (VH)_CDR2 CD3 heavyHGNFGNSYVSWFAY 39 chain (VH)_CDR3 CD3 lightQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRG 31 chain (VL)LIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSN LWVFGGGTKLTVL CD3 lightGSSTGAVTTSNYAN 32 chain_CDR1 CD3 light GTNKRAP 33 chain_CDR2 CD3 lightALWYSNLWV 34 chain_CDR3 pETR12940:QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRG 86 crossedLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSN commonLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF CD3 lightPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY chainICNVNHKPSNTKVDKKVEPKSC (VLCH1) CrossedEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWV 87 CD3 heavySRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVY chainYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLK (VHCκ);SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS e.g. inLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC pCON1057 CD3-CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS 85GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSC CD3-ckappaVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG 88NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

6) Exemplary amino acid sequences of CD3-FolR bispecific antibodies 2+1inverted crossfab formal

Seq ID Description Sequence No VHCH1[9D11]_VHCQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAP  94 [CD3]_Fcknob_PGLALAGQGLEWMGIINPSGGPTSYAQKFQGRVTMTRDTSTSTVYME pCON1057LSSLRSEDTAVYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_Fchole_PGLALA_HYRFQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAP  95GQGLEWMGIINPSGGPTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_LCDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWY  96 pCON1063LQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMNRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC VLCH1[CD3]QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK  86 pETR12940PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSC CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW 428NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNIKVDKKVEPKSCDVHCH1[36F2]_VHCL QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAP 393[CD3]_Fcknob_PGLALA  GQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYME pCON1056LSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 36F2-Fc hole PGLALAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAP 394 pCON1050GQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 36F2 LC pCON1062EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP 395GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYTNEHYYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHXGLSS PVIKSENRGEC CD3 VLCH1QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK  86 pETR12940PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSC

7) Exemplary amino acid sequences of CD3-FolR bispecific antibodies withcommon light chain

VHCH1[16D5]_VHCH1[CD3]_Fcknob EVQLVESGGGLVKPGGSLRLSCAASGFTFSN  89pCON999 AWMSWVRQAPGKGLEWVGRIKSKTDGGITDY AAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQP GGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDD SKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK VHCH1[16D5]_Fchole pCON983EVQLVESGGGLVKPGGSLRLSCAASGFTFSN  90 AWMSWVRQAPGKGLEWVGRIKSKTDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDT AVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK AKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK CD3_common light chain pETR13197QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTT  35 SNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALW YSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS VHCH1[CD3]_VHCH1[16D5]_Fcknob_PGLALAEVQLLESGGGLVQPGGSLRLSCAASGFTFST  91 pETR13932YAMNWVRQAPGKGLEWVSRIRSKYNNYATYY ADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGG GLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFT ISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK CD3_Fcknob_PGLALA pETR13917EVQLLESGGGLVQPGGSLRLSCAASGFTFST  92 YAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDT AVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc_hole_PGLALA_HYRF pETR10755DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL  93 MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFT QKSLSLSPGKVHCL[CD3]_Fcknob_PGLALA pETR13378 EVQLLESGGGLVQPGGSLRLSCAASGFTFST  98YAMNWVRQAPGKGLEWVSRIRSKYNNYATYY ADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS SASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTK NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK16D5 inverted 2 + 1 with N100A in EVQLVESGGGLVKPGGSLRLSCAASGFTFSN  99CDR H3 pETR14096 AWMSWVRQAPGKGLEWVGRIKSKIDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDT AVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGL EWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGAS YVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK16D5 inverted 2 + 1 with S100aA in EVQLVESGGGLVKPGGSLRLSCAASGFTFSN 100CDR H3 pETR14097 AWMSWVRQAPGKGLEWVGRIKSKIDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDT AVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGL EWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNA YVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKCD3 light chain fused to CH1; QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTT 101Fc_PGLALA; pETR13862 SNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALW YSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK16D5 VH fused to constant kappa EVQLVESGGGLVKPGGSLRLSCAASGFTFSN 102chain; pETR13859 AWMSWVRQAPGKGLEWVGRIKSKIDGGITDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDT AVYYCTTPWEWSWYDYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGECCD3 VH fused to constant lambda  EVQLLESGGGLVQPGGSLRLSCAASGFTFST 103chain; pETR13860 YAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDT AVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASPKAAPSVTLFPPSSEELQANKATLVCLI SDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECSIGHV1-46*01(X92343), plus JH4  QVQLVQSGAEVKKPGASVKVSCKASGYTFTS 104element YYMHWVRQAPGQGLEWMGIINPSGGSTSYAQ KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSGGSFDYWGQGTLVTVSS IGHV1-69*06(L22583), plus JH6QVQLVQSGAEVKKPGSSVKVSCKASGGTFSS 105 elementYAISWVRQAPGQGLEWMGGIIPIFGTANYAQ KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGSGGSMDAWGQGTTVTVSS IGHV3-15*01(X92216), plus JH4EVQLVESGGGLVKPGGSLRLSCAASGFTFSN 106 elementAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDY AAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGGSGGSFDYWGQGTLVTVSS IGHV3-23*01(M99660), plus JH4EVQLLESGGGLVQPGGSLRLSCAASGFTFSS 107 elementYAMSWVRQAPGKGLEWVSAISGSGGSTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGSGGSPDYWGQGTLVTVSS IGHV4-59*01(AB019438), plus JH4QVQLQESGPGLVKPSETLSLTCTVSGGSISS 108 elementYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPS LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGGSGGSFDYWGQGTLVTVSS IGHV5-51*01(M99686), plus JH4EVQLVQSGAEVKKPGESLKISCKGSGYSFTS 109 elementYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGSGGSFDYWGQGTLVTVSS CD3 specific antibody based onQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTT 110 humanized CH2527 light chainSNYANWVQEKPGQAFRGLIGGTNKRAPGTPA RFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL hVK1-39(JK4 J-element) DIQMTQSPSSLSASVGDRVTITCRASQSISS111 YLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK VL7_46-13(humanized anti-CD3QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTT 112 antibody light chain)SNYANWVQEKPGQAFRGLIGGTNKRAPGTPA RFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL

8) Exemplary 16D5 variants with reduced affinity

-   -   a. Exemplary light chain variants with reduced affinity

Seq ID Name Sequence No K53A QTVVTQEPSLTVSPGGTVTLTC GSSTGAVTTSNYANWVQQKPGQAPRGLIG GTNARA 113 aa P GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWV FGGGTKLTVL K53A_VL_CDR1 GSSTGAVTTSNYAN  32 K53A_VL_CDR2GTNARAP 396 K53A_VL_CDR3 ALWYSNLWV  34 S93A QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYAN WVQQKPGQAPRGLI GGTNKRA 114 aa PGTPARFSGSLLGGKAALTLSGVQPEDEAEYYC ALWYANLWV FGGGTKLTVL S93A_VL_CDR1GSSTGAVTTSNYAN  32 S93A_VL_CDR2 GTNKRAP  33 S93A_VL_CDR3 ALWYANLWV 397

-   -   b. Exemplary heavy chain variants with reduced affinity

Seq ID Name Sequence No S35H EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMHWVRQAPGKGLEWVG RIKSKTDG 115 aa GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PWEWSWYDY WGQGT LVTVSSAS S35H_VH_CDR1NAWMH 398 S35H_VH_CDR2 RIKSKTDGGTTDYAAPVKG  17 S35H_VH_CDR3 PWEWSWYDY 18 G49S EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMS WVRQAPGKGLEWVS RIKSKTDG116 aa GTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PWEWSWYDY WGQGTLVTVSSAS G49S_VH_CDR1 NAWMS  16 G49S_VH_CDR2 RIKSKTDGGTTDYAAPVKG  17G49S_VH_CDR3 PWEWSWYDY  18 R50S EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMSWVRQAPGKGLEWVG SIKSKTDG 117 aa GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PWEWSWYDY WGQGT LVTVSSAS R50S_VH_CDR1NAWMS  16 R50S_VH_CDR2 SIKSKTDGGTTDYAAPVKG 399 RSOS_VH_CDR3 PWEWSWYDY 18 W96Y EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMS WVRQAPGKGLEWVG RIKSKTDG118 aa GTTDYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PYEWSWYDY WGQGTLVTVSSAS W96Y_VH_CDR1 NAWMS  16 W96Y_VH_CDR2 RIKSKTDGGTTDYAAPVKG  17W96Y_VH_CDR3 PYEWSWYDY 400 W98Y EVQLVESGGGLVKPGGSLRLSCAASGFTFS NAWMSWVRQAPGKGLEWVG RIKSKTDG 119 aa GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT PWEYSWYDY WGQGT LVTVSSAS W98Y_VH_CDR1NAWMS  16 W98Y_VH_CDR2 RIKSKTDGGTTDYAAPVKG  17 W98Y_VH_CDR3 PWEYSWYDY232

9) Additional exemplary embodiments generated from a phage displaylibrary (CDRs underlined)

Seq ID Name Sequence No 90D7 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMHWVRQAPGQGLEWMG IINPSGGS 120 aa TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYTIVVSPFDY WGQGT LVTVSSAS 90D7_VH_CDR1SYYMH   8 90D7_VH_CDR2 IINPSGGSTSYAQKFQG   9 90D7_VH_CDR3 NYTIVVSPFDY233 90C1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG IINPSGGS121 aa TSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR NYFIGSVAMDY WGQGTLVTVSSAS 90C1_VH_CDR1 SYYMH   8 90C1_VH_CDR2 IINPSGGSTSYAQKFQG   990C1_VH_CDR3 NYFIGSVAMDY 234 5E8 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMHWVRQAPGQGLEWMG IINPSGGS 122 aa TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GLTYSMDY WGQGTLVT VSSAS 5E8_VH_CDR1SYYMH   8 5E8_VH_CDR2 IINPSGGSTSYAQKFQG   9 5E8_VH_CDR3 GLTYSMDY 2355E8 VL DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGS 123aa NRAS GVFDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQIPNT FGQGTKVEIKRT5E8_VL_CDR1 RSSQSLLHSNGYNYLD  59 5E8_VL_CDR2 LGSNRAS  60 5E8_VL_CDR3MQALQIPNT 236 12A4 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQAPGKGLEWVS AISGSGGS 124 aa TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK YAYALDY WGQGTLVTV SSAS 12A4_VH_CDR1SYAMS  79 12A4_VH_CDR2 AISGSGGSTYYADSVKG  80 12A4_VH_CDR3 YAYALDY 23712A4 VL EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT 125aa GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQHGSSST FGQGTKVEIKRT 12A4_VL_CDR1RASQSVSSSYLA  52 12A4_VL_CDR2 GASSRAT  53 12A4_VL_CDR3 QQHGSSST 2387A3 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG IINPSGGS 126aa TSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GDFSAGRLMDY WGQGT LVTVSSAS7A3_VH_CDR1 SYYMH   8 7A3_VH_CDR2 IINPSGGSTSYAQKFQG   9 7A3_VH_CDR3GDFSAGRLMDY 239 7A3 VL DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGS 127 aa NRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPIT FGQGTKVEIKR T 7A3_VL_CDR1 RSSQSLLHSNGYNYLD  59 7A3_VL_CDR2LGSNRAS  60 7A3_VL_CDR3 MQALQTPPIT 240 6E10 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG IINPSGGS 128 aaTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GDYNAFDY WGHGTLVT VSSAS6E10_VH_CDR1 SYYMH   8 6E10_VH_CDR2 IINPSGGSTSYAQKFQG   9 6E10_VH_CDR3GDYNAFDY 241 6E10 VL DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGS 129 aa NRAS GVDDRESGSGSGTDFTLKISRVEAEDVGVYYCMQAWHSPT FGQGTKVEIKRT 6E10_VL_CDR1 RSSQSLLHSNGYNYLD  59 6E10_VL_CDR2LGSNRAS  60 6E10_VL_CDR3 MQAWHSPT 242 12F9 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFT SYYMH WVRQAPGQGLEWMG IINPSGGS 130 aaTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GATYTMDY WGQGTLVT VSSAS12F9_VH_CDR1 SYYMH   8 12F9_VH_CDR2 IINPSGGSTSYAQKFQG   9 12F9_VH_CDR3GATYTMDY 243 12F9 VL DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGS 131 aa NRAS GVDDRESGSGSGTDETLKISRVEAEDVGVYYCMQALQTPIT FGQGTKVEIKRT 12F9_VL_CDR1 RSSQSLLHSNGYNYLD  59 12F9_VL_CDR2LGSNRAS  60 12F9_VL_CDR3 MQALQTPIT 244

10) 9D11 Glycosite variants: variable light chain of exemplaryembodiments (CDRs underlined)

Seq ID Variant Sequence No N95SDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGS 132NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMSRTFGQGTKVEIK 12F9_VL_CDR1RSSQSLLHSNGYNYLD  59 12F9_VL_CDR2 LGSNRAS  60 12F9_VL_CDR3 MQASIMSRT  63N95Q DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGS 133NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMQRTFGQGTKVEIK N95Q_VL_CDR1RSSQSLLHSNGYNYLD  59 N95Q_VL_CDR2 LGSNRAS  60 N95Q_VL_CDR3 MQASIMQRT  65T97A DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGS 134NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMNRA FGQGTKVEIK T97A_VL_CDR1RSSQSLLHSNGYNYLD  59 T97A_VL_CDR2 LGSNRAS  60 T97A_VL_CDR3 MQASIMNRA  67T97N DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGS 135NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMNRN FGQGTKVEIK T97N_VL_CDR1RSSQSLLHSNGYNYLD  59 T97N_VL_CDR2 LGSNRAS  60 T97N_VL_CDR3 MQASIMNRN  69

11) Deamination Variants

Seq ID Variant Sequence No 16D5 VH_D52dEEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTEG 248GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGT LVTVSS16D5 VH_D52dQ EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTQG249 GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGT LVTVSSCD3_VH N100A EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNN250 YATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSS CD3_VH S100aAEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNN 251YATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAY WGQGTLVTVSS16D5 [VHCH1]- EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDG252 CD3[VHCH1- GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTN100A]- LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHFcknob_PGLALA TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK16D5-Fchole- EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDG253 PGLALA GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK CD3-CLCQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRA 254PGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDG 255 [VHCH1]-GTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGT CD3[VHCH1-LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH S100aA]-TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGG Fcknob_PGLALASGG GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 9D11QVQLVQSGAEVKKPGASVKVSCHASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGP 256 [VHCH1]-TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWLDYWGQGTLVT CD3[VHCL-VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP N100A]-AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGG Fcknob_PGLALAGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGASYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK9D11-Fchole QVQLVQSGAEVKKPGASVKVSCHASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGP257 TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9D11_LCDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGS 258 [N95Q]NRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASIMQRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CD3_VLCH1QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRA 259PGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 9D11QVQLVQSGAEVKKPGASVKVSCHASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGP 260 [VHCH1]-TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDFAWLDYWGQGTLVT CD3[VHCH1-VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP S100aA]-AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGG Fcknob_PGLALAGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNAYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

12) Mov19 based FolR1 TCBs pf exemplary embodiments (CDRs underlined)

Seq ID Name Sequence No pETR116 QVQLQQSGAELVKPGASVKISCKASGYSFT GYFMN W136 46 VKQSHGKSLEWIG RIHPYDGDTFYNQNFKD KATLTV Mov19DKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMDY W VH-CH1-GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG FcholeCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL PG/LALAYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK pETR116QVQLQQSGAELVKPGASVKISCKASGYSFT GYFMN W 137 47 VKQSHGKSLEWIGRIHPYDGDTFYNQNFKD KATLTV Mov19 DKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMDY WVH-CH1- GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CD3 VH-CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL CL-YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV FcknobEPKSCDGGGGSGGGGSEVQLVESGGGLVQPKGSLKL PG/LALA SCAAS GFTFNTYAMNWVRQAPGKGLEWVA RIRSKYN NYATYYADSVKD RFTISRDDSQSILYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAY WGQGTLVTVSAASV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK pETR116DIELTQSPASLAVSLGQRAIISC KASQSVSFAGTSL 138 44 MH WYHQKPGQQPKLLIY RASNLEAGVPTRFSGSGSK Mov19 LC TDFTLNIHPVEEEDAATYYC QQSREYPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

Seq Name Sequence ID No Hu IgG1 DKTHTCPPCPAPELLGGPSVFLFPPKPKDT 245 FcLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

13) Additional FolR1 TCBs with intermediate affinity binders (CDRsaccording to Kabat, underlined)

Seq ID Name Sequence No 16D5 variant EVQLVESGGGLVKPGGSLRLSCAASGFTFS 401W96Y/D5 NAWMSWVRQAPGKGLEWVGRIKSKTEGGTT 2E VHDYAAPVKGRFTISRDDSKNTLYLQMNSLKT EDTAVYYCTTPYEWSWYDYWGQGTLVTVSS W96Y/D5NAWMS  16 2E_VH CDR1 W96Y/D5 RIKSKTEGGTTDYAAPVKG 402 2E_VH CDR2 W96Y/D5PYEWSWYDY 400 2E_VH CDR3 16D5 variant QAVVTQEPSLTVSPGGTVTLTCGSSTGAVT  31W96Y/D5 TSNYANWVQEKPGQAFRGLIGGTNKRAPGT 2E VLPARFSGSLLGGKAALTLSGAQPEDEAEYYC ALWYSNLWVFGGGTKLTVL W96Y/D5 2E_CD3-EVQLVESGGGLVKPGGSLRLSCAASGFTFS 403 VHCH1_Fc-knob_PGLALANAWMSWVRQAPGRGLEWVGRIKSKTEGGTT pETR14945 DYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLE SGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSV KGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDEL TKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKW96Y/D5 2E_Fc- EVQLVESGGGLVKPGGSLRLSCAASGFTFS 404 hole_PGLALA_HYRFNAWMSWVRQAPGKGLEWVGRIKSKTEGGTT pETR14946 DYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPYEWSWYDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDE LTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNRFTQKSLSLSPGK 14B1 VHEVQLLESGGGLVQPGGSLRLSCAASGFTFS 405 SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAED TAVYYCAR GDYRYRYFDY WGQGTLVTVSS 14B1 VLSSELTQDPAVSVALGQTVRITCQGDSLRSY 406 YASWYQQKPGQAPVLVIYGKENRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSR ESPPTGLVVFGGGTKLTVL14B1[EE]_CD3[VLCH1]_Fc- EVQLLESGGGLVQPGGSLRLSCAASGFTFS 407 knob_PGLALASYAMSWVRQAPGKGLEWVSAISGSGGSTYY pETR14976 ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRYRYFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQE PSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGS LLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKREEQYNSTYRVVSVLTVLHQDWL NPGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 14B1[EE]_Fc-hole_PGLALAEVQLLESGGGLVQPGGSLRLSCAASGFTFS 408 pETR14977SYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDYRYRYFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDEL TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK14B1 LC [KK] Constant  SSELTQDPAVSVALGQTVRITCQGDSLRSY 409 lambdaYASWYQQKPGQAPVLVIYGKNNRPSGIPDR pETR14979 FSGSSSGNTASLTITGAQAEDEADYYCNSRESPPTGLVVFGGGTKLTVLGQPKAAPSVTL FPPSSKKLQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSY LSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS9C7 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFT 410 SYYMH WVRQAPGQGLEWMGIINPSGGSTSY AQKFQG RVTMTRDTSTSTVYMELSSLRSED TAVYYCAR GDWSYYMDYWGQGTLVTVSS 9C7 VL DIVMTQSPLSLPVTPGEPASISC RSSQSLL 411 HSNGYNYLDWYLQKPGQSPQLLIY LGSNRA S GVPDRFSGSGSGTDFTLKISRVEAEDVGV YYC MQARQTPTFGQGTKVEIK 9C7[EE]_CD3[VLCH1]_Fc- QVQLVQSGAEVKKPGASVKVSCKASGYTFT 412knob_PGLALA SYYMHWVRQAPGQGLEWMGIINPSGGSTSY pETR14974AQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARGDWSYYMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWV QEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW VFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9C7[EE]_Fc-hole_PGLALA QVQLVQSGAEVKKPGASVKVSCKASGYTFT 413pETR14975 SYYMHWVRQAPGQGLEWMGIINPSGGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWSYYMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELT KNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK9C7 LC [RK] pETR14980 DIVMTQSPLSLPVTPGEPASISCRSSQSLL 414HSNGYNYLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPTFGQGTKVEIKRTVAAPSVF IFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

14) Antigen Sequences

Seq ID Antigen Sequence No hu MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELL 139FolR1 NVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQ DTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCA VGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGP WAAWPFLLSLALMLLWLLS huFolR1RIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCR 140 ECD-PWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEM AcTev-APACKRHFIQDTCLYECSPNLGPWIQQVDQSWRK Fcknob-ERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGW Avi tagNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWT HSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMVDEQLYFQGGSPKSADKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKSGGLNDIFEAQKIEWHE FcholeDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS 141RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRD ELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS CSVMHEALHNRFTQKSLSLSPGK muMAHLMTVQLLLLVMWMAECAQSRATRARTELLNV 142 FolR1CMDAKHHKEKPGPEDNLHDQCSPWKTNSCCSTNT SQEAHKDISYLYRFNWNHCGTMTSECKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERILDVPLCKED CQQWWEDCQSSFTCKSNWHKGWNWSSGHNECPVGASCHPFTFYFPTSAALCEEIWSHSYKLSNYSRGS GRCIQMWFDPAQGNPNEEVARFYAEAMSGAGLHGTWPLLCSLSLVLLWVIS mu TRARTELLNVCMDAKHHKEKPGPEDNLHDQCSPW 143 FolR1KTNSCCSTNTSQEAHKDISYLYRFNWNHCGTMTS ECD-ECKRHFIQDTCLYECSPNLGPWIQQVDQSWRKER AcTev-ILDVPLCKEDCQQWWEDCQSSFTCKSNWHKGWNW Fcknob-SSGHNECPVGASCHPFTFYFPTSAALCEEIWSHS AvitagYKLSNYSRGSGRCIQMWFDPAQGNPNEEVARFYA EAMVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE cy FolR1 MAQRMTTQLLLLLVWVAVVGEAQTRTARARTELL 144NVCMNAKHHKEKPGPEDKLHEQCRPWKKNACCST NTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCK EDCERWWEDCRTSYTCKSNWHKGWNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYKVSNYSR GSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPLLLSLALTLLWLLS cy FolR1 RTARARTELLNVCMNAKHHKEKPGPEDKLHEQCR 145 ECD-PWKKNACCSTNTSQEAHKDVSYLYRFNWNHCGEM AcTev-APACKRHFIQDTCLYECSPNLGPWIQQVDQSWRK Fcknob-ERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGW Avi tagNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWT YSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMVDEQLYFQGGSPKSADKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKSGGLNDIFEAQKIEWHE huMVWKWMPLLLLLVCVATMCSAQDRTDLLNVCMDA 146 FolR2KHHKTKPGPEDKLHDQCSPWKKNACCTASTSQEL HKDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKERFLDVPLCKEDCQRW WEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSGRCI QMWFDSAQGNPNEEVARFYAAAMHVNAGEMLHGTGGLLLSLALMLQLWLLG hu TMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQ 147 FolR2CSPWKKNACCTASTSQELHKDTSRLYNFNWDHCG ECD-KMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSW AcTev-RKERFLDVPLCKEDCQRWWEDCHTSHTCKSNWHR Fcknob-GWDWTSGVNKCPAGALCRTFESYFPTPAALCEGL Avi tagWSHSYKVSNYSRGSGRCIQMWFDSAQGNPNEEVA RFYAAAMHVVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE hu MAWQMMQLLLLALVTAAGSAQPRSARARTDLLNV 148FolR3 CMNAKHHKTQPSPEDELYGQCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPTCKRHFIQDS CLYECSPNLGPWIRQVNQSWRKERILNVPLCKEDCERWWEDCRTSYTCKSNWHKGWNWTSGINECPAG ALCSTFESYFPTPAALCEGLWSHSFKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFYAAAMNAGAPSR GIIDS huSARARTDLLNVCMNAKHHKTQPSPEDELYGQCSP 149 FolR3WKKNACCTASTSQELHKDTSRLYNFNWDHCGKME ECD-PTCKRHFIQDSCLYECSPNLGPWIRQVNQSWRKE AcTev-RILNVPLCKEDCERWWEDCRTSYTCKSNWHKGWN Fcknob-WTSGINECPAGALCSTFESYFPTPAALCEGLWSH Avi tagSFKVSNYSRGSGRCIQMWFDSAQGNPNEEVAKFY AAAMNAGAPSRGIIDSVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIE WHE hu CD3ϵMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQT 150PYKVSISGTTVILTCPQYPGSEILWQHNDKNIGG DEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIV DICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRR I

2) Nucleotide sequences of exemplary embodiments

Seq ID Description Sequence No 16A3CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 151GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACGCTGGTGTTACTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 15A1CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 152GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACATCGGTGTTGTTACTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 18D3CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 153GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACACTGGTGGTTCTTCTGCTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTT TCTTCT 19E5CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGNTTCC 154GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAATGGCGTCGTTACACTTCTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 19A4CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 155GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGGTTGGATCCGTTGGGAACATTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 15H7CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 156GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACCTGTTCTCTACTTCTTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 15E6CAGGTGCAATTGGTTCAATCTGGTGCTGAGGTAAAAAAACCGGGCGCTTCC 157GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACATCGGTATCGTTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 158CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCT TCC 15E12GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 159CNGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACCGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTACTTCGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCT TCC 21D1CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 160GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTACGTTGGTGTTTCTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 16F12CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGNTTCC 161GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCNTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTTCACTGTTCTGCGTGTTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 21A5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 162CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGGCTTGGTTCGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCT TCC 21G8GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 163CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACCGAAGACACCGCAGTCTACTACTGTACTACCCCTTGGGAATGGGCTTACTTCGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCT TCC 19H3CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 164GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCACTGGTTGGTCTCGTTGGGGTTACATGGACTATTGGGGCCAAGGCACCCTCGTAACGGTTTCT TCT 20G6CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 165GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGAATGGATCCGTTACTACCATTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 20H7CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 166GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGTTGGTTGGTACCGTTGGGGTTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCT 11F8_VHCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCG 167GTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTAACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGAGCTGTTTTCTACCGTGCTTGGTACTCTTTCGACTACTGGGGCCAAGGGACCACCGTGACCGTC TCCTCA 11F8_VLGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGAC 168CGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATACCAGCCCACCACCAACGTTTGGCCAGGGCACC AAAGTCGAGATCAAG36F2_VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 169GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 36F2_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 170AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTCGGCCAGGGGACCAAAGTGGAAATCAAA 9D11_VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 171GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 172CCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 173 N95SCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAGCCGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 174 N95QCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 175 T97ACCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGGCTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 9D11_VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 176 T97NCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGAATTTTGGTCAAGGCACCAAGGTCGAAATTAAA 5D9_VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 177GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTACATCGACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 5D9_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 178AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGATAACTGGAGCCCAACGTTCGGCCAGGGGACC AAAGTGGAAATCAAA6B6_VH CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 179GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTACGTTGACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 6B6_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 180AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACCTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGATATTTGGAGCCCAACGTTCGGCCAGGGGACC AAAGTGGAAATCAAA14E4_VH GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC 181CTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACTCTTCTTACGTTGAATGGTACGCTTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTC TCGAGT 14E4_VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 182AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTCCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGCCAACCAGCAGCCCAATTACGTTCGGCCAGGGG ACCAAAGTGGAAATCAAACD3 heavy chain (VHCH1)GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCT 183CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGC Crossed CD3 heavy chain (VHCκ)GAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCT 184CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT Mutagenesis primer GAB7734 N95QGCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGG 185 Mutagenesis primer GAB7735 N95SCAGGCAAGCATTATGAGCCGGACTTTTGGTCAAGG 186 Mutagenesis primer GAB7736 T97ACATTATGAACCGGGCTTTTGGTCAAGGCACCAAGGTC 187Mutagenesis primer GAB7737 T97N CATTATGAACCGGAATTTTGGTCAAGGCACCAAGGTC188 VHCH1[16D5]_VHCH1[CD3]_Fcknob_PGLALAGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 189pCON999 (Inverted TCB with 16D5 2 + CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATG1: pCON999 + pCON983 + pETR13197)AGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[16D5]_Fchole_PGLALA_HYRFGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 190 pCON983CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD3_common light chain pETR13197CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 191GTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC VHCH1[CD3]_VHCH1[16D5]_Fcknob_PGLALAGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCT 192pETR13932 (Classical TCB with 16D5; CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATG2 + 1: pETR13932 + pCON983 + AACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATC pETR13197)AGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA VHCH1[CD3]_Fcknob_PGLALAGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCT 193pETR13719 (16D5 IgG format, 1 + 1:CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGpETR13719 + pCON983 + pETR13197)AACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Fc_hole_PGLALA_HYRFGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA 194pETR10755 (16D5 Head-to-tail, 1 + 1:CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCpCON999 + pETR10755 + pETR13197)CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAAVHCH1[9D11]_VHCL[CD3]1_Fcknob_PGLALACAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 195pCON1057 (9D11 inverted format, 2 + GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATG1: pCON1057 + pCON1051 + pCON1063 + CACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATT pETR12940)AACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11_Fchole_PGLALA_HYRFCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 196 pCON1051GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA9D11_LC GATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 197 pCON1063CCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGAACCGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT VLCH1[CD3]CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 198 pETR12940GTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT VHCL[CD3]_Fcknob_PGLALAGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCT 199pETR13378 (9D11 CrossMab format,  CTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATG1 + 1: pETR13378 + pCON1051 + AACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCpCON1063 + pETR12940)AGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA16D5 inverted 2 + 1 with N100A inGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 200 CDR H3 pETR14096CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATG(pETR14096 + pCON983 + pETR13197)AGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5 inverted 2 + 1 with S100aA in GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 201CDR H3 pETR14097 (pETR14097 + CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATG pCON983 + pETR13197)AGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD3 light chain fused to CH1;CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 202Fc_PGLALA; pETR13862 (Kappa-lambdaGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACantibody with CD3 common light chainGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCfused to CH1 + Fc_PGLALA.GGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGVHs fused to kappa or lambda CTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGconstant chain pETR13859 + GCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGApETR13860 + pETR13862)GGCACCAAGCTGACAGTGCTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG  GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA16D5 VH fused to constant kappa GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCC 203 chain; pETR13859CTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCT TTCAACCGGGGCGAGTGCCD3 VH fused to constant lambda GAAGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGGCGGATCT 204 chain; pETR13860CTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCCACCTACGCCATGAACTGGGTGCGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGTCCCGGATCAGATCCAAGTACAACAACTACGCCACCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCTCGGGACGACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACTCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCCCCAAGGCTGCCCCCAGCGTGACCCTGTTTCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC VHCH1[36F2]_VHCL[CD3]_Fcknob_PGLALACAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 246 pCON1056GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 36F2-Fc hole PGLALACAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 247 pCON1050GTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 36F2 LCGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA  97 pCON1062AGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCANGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT CD3 VLCH1CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 198 pETR12940GTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCTTGT Seq ID Name Sequence No K53ACAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 205 ntGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACGCCAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTA S93ACAGACCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACC 206 ntGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACGCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTA

Seq Name Sequence ID No S35HGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 207 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGCACTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCTGCTAGC G49SGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 208 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGTCCCGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCTGCTAGC R50SGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 209 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGATCTATCAAGAGCAAGACCGACGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTGGTCTTGGTACGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCC TCT GCTAGC W96YGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 210 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCT GCTAGC W98YGAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCT 211 ntCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGCTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAGACCGATGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTGGGAGTACTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCA TCT GCTAGC

Seq Name Sequence ID No 90D7CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 212 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACACTATCGTTGTTTCTCCGTTCGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 90C1CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 213 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCAACTACTTCATCGGTTCTGTTGCTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 5E8 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 214 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTCTGACTTACTCTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGC 5E8 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 215 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGATTCCAAACACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 12A4 VHGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC 216 ntCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATACGCTTACGCTCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGC 12A4 VLGAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAA 217 ntAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGCATGGCAGCAGCAGCACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACG 7A3 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 218 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCTCTGCTGGTCGTCTGATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCT TCTGCTAGC 7A3 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 219 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCACCAATTACCTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 6E10 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 220 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTACAACGCTTTCGACTATTGGGGTCACGGCACCCTCGTAACGGTTTCTTCTGCTAGC 6E10 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 221 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCATGGCATAGCCCAACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG 12F9 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCC 222 ntGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGCTACTTACACTATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGC 12F9 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAA 223 ntCCGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACTGCAGACCCCAATTACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACG

Seq Name Sequence ID No pETR11646CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTCC 224 Mov19 VH-GTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCATG CH1-FcholeAACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGAATC PG/LALACACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGCTGAGCCTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGACGGCAGCCGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAApETR11647 CAGGTGCAGCTGCAGCAGTCTGGCGCCGAGCTCGTGAAACCTGGCGCCTCC 225Mov19 VH- GTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACTTCATG CH1-CD3AACTGGGTCAAGCAGAGCCACGGCAAGAGCCTGGAATGGATCGGCAGAATC VH-CL-CACCCCTACGACGGCGACACCTTCTACAACCAGAACTTCAAGGACAAGGCC FcknobACCCTGACCGTGGACAAGAGCAGCAACACCGCCCACATGGAACTGCTGAGC PG/LALACTGACCAGCGAGGACTTCGCCGTGTACTACTGCACCAGATACGACGGCAGCCGGGCCATGGATTATTGGGGCCAGGGCACCACCGTGACAGTGTCCAGCGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAAGTGCAGCTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCTAAGGGCTCTCTGAAGCTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGGCCCGGATCAGAAGCAAGTACAACAATTACGCCACCTACTACGCCGACAGCGTGAAGGACCGGTTCACCATCAGCCGGGACGACAGCCAGAGCATCCTGTACCTGCAGATGAACAACCTGAAAACCGAGGACACCGCCATGTACTACTGCGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTGCTGCTAGCGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA pETR11644GACATCGAGCTGACCCAGAGCCCTGCCTCTCTGGCCGTGTCTCTGGGACAG 226 Mov19 LCAGAGCCATCATCAGCTGCAAGGCCAGCCAGAGCGTGTCCTTTGCCGGCACCTCTCTGATGCACTGGTATCACCAGAAGCCCGGCCAGCAGCCCAAGCTGCTGATCTACAGAGCCAGCAACCTGGAAGCCGGCGTGCCCACAAGATTTTCCGGCAGCGGCAGCAAGACCGACTTCACCCTGAACATCCACCCCGTGGAAGAAGAGGACGCCGCCACCTACTACTGCCAGCAGAGCAGAGAGTACCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

Seq Variant Sequence ID No 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 261 VH_D52dETGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGAGGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 262 VH_D52dQTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTCAGGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCC CD3_VHGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTC 263 N100ATGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA CCGTGTCAAGC CD3_VHGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTC 264 S100aATGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA CCGTGTCAAGC 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 265 [VHCH1]-CD3TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG [VHCH1-N100A]-CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG Fcknob_PGLALATCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 16D5-Fchole-GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 266 PGLALATGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD3-CLCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCG 267TGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCC 268 [VHCH1]-CD3TGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAG [VHCH1-S100aA]-CTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAG Fcknob_PGLALATCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 269 [VHCH1]-CD3TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA [VHCL-N100A]-CTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAAC Fcknob_PGLALACCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCGCCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11-FcholeCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 270TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9D11_LCGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAAC 271 [N95Q]CGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCAAGCATTATGCAGCGGACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT CD3_VLCH1CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCG 272TGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAG GTGGAACCCAAGTCTTGT9D11 CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 273[VHCH1]-CD3 TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA[VHCH1-S100aA]- CTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACFcknob_PGLALA CCAAGCGGTGGCCCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTTCGCTTGGCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACGCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Seq Name Sequence ID No 16D5GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTC 415 variantTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAG W96Y/CTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAG D52EAGCAAGACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGT VHTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCT W96Y/GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTC 416D52E-_CD3-VHCH1_Fc- TGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAGknob_PGLALA CTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGpETR14945 AGCAAGACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA W96Y/GAGGTGCAATTGGTGGAAAGCGGAGGCGGCCTCGTGAAGCCTGGCGGATCTC 417D52E-_Fc-hole_PGLALA_HYRFTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAACGCCTGGATGAG pETR14946CTGGGTGCGCCAGGCCCCTGGAAAAGGACTCGAGTGGGTGGGACGGATCAAGAGCAAGACCGAGGGCGGCACCACCGACTATGCCGCCCCTGTGAAGGGCCGGTTCACCATCAGCAGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGCACCACCCCCTACGAGTGGTCTTGGTACGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTC TCCGGGTAAA 14B1GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCC 418 VHTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT 14B1TCTTCTGAACTGACTCAAGATCCAGCTGTTAGCGTGGCTCTGGGTCAGACTG 419 VLTACGTATCACCTGCCAAGGCGATTCTCTGCGCTCCTACTACGCAAGCTGGTACCAGCAGAAACCGGGTCAGGCCCCAGTTCTGGTGATTTACGGCAAAAACAACCGTCCGTCTGGGATCCCGGACCGTTTCTCCGGCAGCTCTTCCGGTAACACGGCGAGCCTCACCATCACTGGCGCTCAAGCAGAAGACGAGGCCGACTATTACTGTAACTCTCGGGAAAGCCCACCAACCGGCCTGGTTGTCTTCGGTGGCGGTACC AAGCTGACCGTCCTA14B1 GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCC 420 [EE]_CD3TGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAG[VLCH1]_Fc-knob_PGLALACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGT pETR14976GGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACTGCCGCTCTGGGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACGAGAAGGTGGAACCCAAGTCCTGCGACGGTGGCGGAGGTTCCGGAGGCGGAGGATCCCAGGCTGTCGTGACCCAGGAACCCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACCTGTGGATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCCCTGACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCCCTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACCCACACCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 14B1GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCC 421[EE]_Fc-hole_PGLALA TGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGpETR14977 CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGCGTGGTGACTACCGTTACCGTTACTTCGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 14B1 LCTCTTCTGAACTGACTCAAGATCCAGCTGTTAGCGTGGCTCTGGGTCAGACTG 422 [KK]TACGTATCACCTGCCAAGGCGATTCTCTGCGCTCCTACTACGCAAGCTGGTA ConstantCCAGCAGAAACCGGGTCAGGCCCCAGTTCTGGTGATTTACGGCAAAAACAAC lambdaCGTCCGTCTGGGATCCCGGACCGTTTCTCCGGCAGCTCTTCCGGTAACACGG pETR14979CGAGCCTCACCATCACTGGCGCTCAAGCAGAAGACGAGGCCGACTATTACTGTAACTCTCGGGAAAGCCCACCAACCGGCCTGGTTGTCTTCGGTGGCGGTACCAAGCTGACCGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCAAGAAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC 9C7 VHCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 423TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCT 9C7 VLGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAAC 424CGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTACAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACGGCAGACCCCAACTTTTGGTCAAGGCACCAAGGTCGAAATTAAA 9C7[EE]_CD3CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 425 [VLCH1]_FcknobTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCA PGLALACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAAC pETR14974CCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACTGCCGCTCTGGGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACGAGAAGGTGGAACCCAAGTCCTGCGACGGTGGCGGAGGTTCCGGAGGCGGAGGATCCCAGGCTGTCGTGACCCAGGAACCCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACCTGTGGATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCCCTGACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCCCTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACCCACACCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9C7[EE]_Fc-hole_PGLALA CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCG 426 pETR14975TTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCGTGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCGGTGACTGGTCTTACTACATGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 9C7 LCGATATTGTTATGACTCAATCTCCACTGTCTCTGCCGGTGACTCCAGGCGAAC 427 [RK]CGGCGAGCATTTCTTGCCGTTCCAGCCAGTCTCTGCTGCACTCCAACGGCTA pETR14980CAACTATCTCGATTGGTACCTGCAAAAACCGGGTCAGAGCCCTCAGCTGCTGATCTACCTGGGCTCTAACCGCGCTTCCGGTGTACCGGACCGTTTCAGCGGCTCTGGATCCGGCACCGATTTCACGTTGAAAATCAGCCGTGTTGAAGCAGAAGACGTGGGCGTTTATTACTGTATGCAGGCACGGCAGACCCCAACTTTTGGTCAAGGCACCAAGGTCGAAATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTExemplary Anti-PD1 Antagonist Sequences

Seq Description Sequence ID No anti-PDL1QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLE 274 antibodyWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK anti-PDL1EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL 275 antibodyLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC anti-PDL1QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLE 276 antibodyWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK anti-PDL1EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQ 277 antibodyAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC heavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLE 278WVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG lightDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKL 279LIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC anti-PDL1EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLE 280 antibodyWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VHVYYCARRHWPGGFDYWGQGTLVTVSS anti-PDL1EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLE 281 antibodyWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VHVYYCARRHWPGGFDYWGQGTLVTVSSASTK anti-PDL1DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKL 282 antibodyLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYL VL YHPATFGQGTKVEIKRHVR-H1 GFTFSX1SWIH 283 HVR-H2 AWIX2PYGGSX3YYADSVKG 284 HVR-H3 RHWPGGFDY285 HVR-L1 RASQX4X5X6TX7X8A 286 HVR-L2 SASX9LX10S 287 HVR-L3QQX11X12X13X14PX15T 288 HVR-H1 GFTFSDSWIH 289 HVR-H2 AWISPYGGSTYYADSVKG290 HVR-H3 RHWPGGFDY 291 HVR-L1 RASQDVSTAVA 292 HVR-L2 SASFLYS 293HVR-L3 QQYLYHPAT 294 anti-PDL1 EVQLVESGGGLVQPGGSLRLSCAAS 295 antibodyHC-FR1 anti-PDL1 HC-FR2 is WVRQAPGKGLEWV 296 antibody HC-FR2 anti-PDL1RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR 297 antibody HC-FR3 anti-PDL1WGQGTLVTVSA 298 antibody HC-FR4 anti-PDL1 WGQGTLVTVSS 299 antibodyHC-FR4 LC-FR1 DIQMTQSPSSLSASVGDRVTITC 300 LC-FR2 WYQQKPGKAPKLLIY 301LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 302 LC-FR4 FGQGTKVEIKR 303anti-PDL1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLE 382 antibodyWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VHVYYCARRHWPGGFDYWGQGTLVTVSA anti-PDL1DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKL 383 antibodyLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYL VL YHPATFGQGTKVEIKRExemplary Anti-TIM3 Antibody Sequences

Sequences of exemplary anti-TIM3 antibody amino acid sequences andexemplary TIM3 sequences are set forth in the sequence listing below asfollows:

SEQ ID NO: 304 heavy chain HVR-H1, Tim3_0016 SEQ ID NO: 305heavy chain HVR-H2, Tim3_0016 SEQ ID NO: 306heavy chain HVR-H3, Tim3_0016 SEQ ID NO: 307light chain HVR-L1, Tim3_0016 SEQ ID NO: 308light chain HVR-L2, Tim3_0016 SEQ ID NO: 309light chain HVR-L3, Tim3_0016 SEQ ID NO: 310heavy chain variable domain VH, Tim3_0016 SEQ ID NO: 311light chain variable domain VL, Tim3_0016 SEQ ID NO: 312heavy chain variable domain VH, Tim3_0016 variant (0018) SEQ ID NO: 313light chain variable domain VL, Tim3_0016 variant (0018) SEQ ID NO: 314light chain HVR-L1, Tim3_0016_HVR-L1 variant 1_NQ(removal of glycosylation sity by N to Q mutation) SEQ ID NO: 315light chain HVR-L1, Tim3_0016_HVR-L1 variant 2_NS(removal of glycosylation sity by N to S mutation) SEQ ID NO: 316heavy chain HVR-H1, Tim3_0021 SEQ ID NO: 317heavy chain HVR-H2, Tim3_0021 SEQ ID NO: 318heavy chain HVR-H3, Tim3_0021 SEQ ID NO: 319light chain HVR-L1, Tim3_0021 SEQ ID NO: 320light chain HVR-L2, Tim3_0021 SEQ ID NO: 321light chain HVR-L3, Tim3_0021 SEQ ID NO: 322heavy chain variable domain VH, Tim3_0021 SEQ ID NO: 323light chain variable domain VL, Tim3_0021 SEQ ID NO: 324heavy chain HVR-H1, Tim3_0022 SEQ ID NO: 325heavy chain HVR-H2, Tim3_0022 SEQ ID NO: 326heavy chain HVR-H3, Tim3_0022 SEQ ID NO: 327light chain HVR-L1, Tim3_0022 SEQ ID NO: 328light chain HVR-L2, Tim3_0022 SEQ ID NO: 329light chain HVR-L3, Tim3_0022 SEQ ID NO: 330heavy chain variable domain VH, Tim3_0022 SEQ ID NO: 331light chain variable domain VL, Tim3_0022 SEQ ID NO: 332heavy chain HVR-H1, Tim3_0026 SEQ ID NO: 333heavy chain HVR-H2, Tim3_0026 SEQ ID NO: 334heavy chain HVR-H3, Tim3_0026 SEQ ID NO: 335light chain HVR-L1, Tim3_0026 SEQ ID NO: 336light chain HVR-L2, Tim3_0026 SEQ ID NO: 337light chain HVR-L3, Tim3_0026 SEQ ID NO: 338heavy chain variable domain VH, Tim3_0026 SEQ ID NO: 339light chain variable domain VL, Tim3_0026 SEQ ID NO: 340heavy chain HVR-H1, Tim3_0028 SEQ ID NO: 341heavy chain HVR-H2, Tim3_0028 SEQ ID NO: 342heavy chain HVR-H3, Tim3_0028 SEQ ID NO: 343light chain HVR-L1, Tim3_0028 SEQ ID NO: 344light chain HVR-L2, Tim3_0028 SEQ ID NO: 345light chain HVR-L3, Tim3_0028 SEQ ID NO: 346heavy chain variable domain VH, Tim3_0028 SEQ ID NO: 347light chain variable domain VL, Tim3_0028 SEQ ID NO: 348heavy chain HVR-H1, Tim3_0030 SEQ ID NO: 349heavy chain HVR-H2, Tim3_0030 SEQ ID NO: 350heavy chain HVR-H3, Tim3_0030 SEQ ID NO: 351light chain HVR-L1, Tim3_0030 SEQ ID NO: 352light chain HVR-L2, Tim3_0030 SEQ ID NO: 353light chain HVR-L3, Tim3_0030 SEQ ID NO: 354heavy chain variable domain VH, Tim3_0030 SEQ ID NO: 355light chain variable domain VL, Tim3_0030 SEQ ID NO: 356heavy chain HVR-H1, Tim3_0033 SEQ ID NO: 357heavy chain HVR-H2, Tim3_0033 SEQ ID NO: 358heavy chain HVR-H3, Tim3_0033 SEQ ID NO: 359light chain HVR-L1, Tim3_0033 SEQ ID NO: 360light chain HVR-L2, Tim3_0033 SEQ ID NO: 361light chain HVR-L3, Tim3_0033 SEQ ID NO: 362heavy chain variable domain VH, Tim3_0033 SEQ ID NO: 363light chain variable domain VL, Tim3_0033 SEQ ID NO: 364heavy chain HVR-H1, Tim3_0038 SEQ ID NO: 365heavy chain HVR-H2, Tim3_0038 SEQ ID NO: 366heavy chain HVR-H3, Tim3_0038 SEQ ID NO: 367light chain HVR-L1, Tim3_0038 SEQ ID NO: 368light chain HVR-L2, Tim3_0038 SEQ ID NO: 369light chain HVR-L3, Tim3_0038 SEQ ID NO: 370heavy chain variable domain VH, Tim3_0038 SEQ ID NO: 371light chain variable domain VL, Tim3_0038 SEQ ID NO: 372an exemplary Pseudomonas exotoxin A variant 1 (deimunized PE24 example)SEQ ID NO: 373 an exemplary Pseudomonas exotoxin A variant 2(deimunized PE24 example) SEQ ID NO: 374human kappa light chain constant region SEQ ID NO: 375human lambda light chain constant region SEQ ID NO: 376human heavy chain constant region derived from IgG1 SEQ ID NO: 377human heavy chain constant region derived from IgG1 withmutations L234A and L235A SEQ ID NO: 378human heavy chain constant region derived from IgG1 withmutations L234A, L235A and P329G SEQ ID NO: 379human heavy chain constant region derived from IgG4 SEQ ID NO: 380exemplary human TIM3 sequences SEQ ID NO: 381human TIM3 Extracellular Domain (ECD) <210> 304 <211>   9 <212> PRT<213> Mus musculus <400> 304 Gly Phe Ser Leu Ser Thr Ser Gly Met1               5 <210> 305 <211>   3 <212> PRT <213> Mus musculus<400> 305 Leu Asn Asp 1 <210> 306 <211>   8 <212> PRT <213> Mus musculus<400> 306 Asn Gly Tyr Leu Tyr Ala Leu Asp 1               5 <210> 307<211>   6 <212> PRT <213> Mus musculus <400> 307 Ser Ser Ser Val Asn Tyr1               5 <210> 308 <211>   3 <212> PRT <213> Mus musculus<400> 308 Asp Ala Phe 1 <210> 309 <211>   7 <212> PRT <213> Mus musculus<400> 309 Trp Ser Ser Tyr Pro Trp Thr 1               5 <210> 310<211> 120 <212> PRT <213> Mus musculus <400> 310Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1               5                   10                  15Thr Leu Arg Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser            20                  25                  30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu        35                  40                  45Trp Leu Ala His Ile Trp Leu Asn Asp Asp Val Phe Phe Asn Pro Ala    50                  55                  60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val65                  70                  75                  80Phe Leu Gln Ile Ala Ser Val Val Thr Ala Asp Thr Ala Thr Tyr Tyr                85                  90                  95Cys Val Arg Ala Asn Gly Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly Gln            100                 105                 110Gly Thr Ser Val Thr Val Ser Ser         115                 120<210> 311 <211> 106 <212> PRT <213> Mus musculus <400> 311Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1               5                   10                  15Gln Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Thr            20                  25                  30Gln Trp Tyr Gln Gln Lys Leu Gly Ser Ser Pro Lys Leu Trp Ile Tyr        35                  40                  45Asp Ala Phe Lys Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser    50                  55                  60Gly Thr Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65                  70                  75                  80Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro Trp Thr                85                  90                  95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys            100                 105 <210> 312 <211> 120 <212> PRT<213> Mus musculus <400> 312Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln1               5                   10                  15Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser            20                  25                  30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu        35                  40                  45Trp Leu Ala His Ile Trp Leu Asn Asp Asp Val Phe Phe Asn Pro Ala    50                  55                  60Leu Lys Arg Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val65                  70                  75                  80Phe Leu Gln Ile Ala Ser Val Val Thr Ala Asp Thr Ala Thr Tyr Tyr                85                  90                  95Cys Val Arg Ala Asn Gly Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly Gln            100                 105                 110Gly Ile Ser Val Thr Val Ser Ser         115                 120<210> 313 <211> 106 <212> PRT <213> Mus musculus <400> 313Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1               5                   10                  15Gln Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Thr            20                  25                  30Gln Trp Tyr Gln Gln Lys Leu Gly Ser Ser Pro Lys Leu Trp Ile Tyr        35                  40                  45Asp Ala Phe Lys Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser    50                  55                  60Gly Thr Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65                  70                  75                  80Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro Trp Thr                85                  90                  95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys            100                 105 <210> 314 <211>   6 <212> PRT<213> Mus musculus <400> 314 Ser Ser Ser Val Gln Tyr 1               5<210> 315 <211>   6 <212> PRT <213> Mus musculus <400> 315Ser Ser Ser Val Ser Tyr 1               5 <210> 316 <211>   7 <212> PRT<213> Mus musculus <400> 316 Gly Tyr Ser Phe Thr Ser Tyr1               5 <210> 317 <211>   3 <212> PRT <213> Mus musculus<400> 317 Ser Asp Ser 1 <210> 318 <211>   9 <212> PRT <213> Mus musculus<400> 318 Gly Tyr Tyr Ala Trp Tyr Tyr Phe Asp 1               5<210> 319 <211>   7 <212> PRT <213> Mus musculus <400> 319Ser Gln Ser Ile Gly Asn Asn 1               5 <210> 320 <211>   3<212> PRT <213> Mus musculus <400> 320 Tyr Ala Ser 1 <210> 321 <211>   6<212> PRT <213> Mus musculus <400> 321 Ser Asn Ser Trp Pro Leu1               5 <210> 322 <211> 120 <212> PRT <213> Mus musculus<400> 322Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala1               5                   10                  15Ser Val Gln Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr            20                  25                  30Leu Leu His Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile        35                  40                  45Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe    50                  55                  60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65                  70                  75                  80Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                85                  90                  95Ala Arg Asp Gly Tyr Tyr Ala Trp Tyr Tyr Phe Asp Cys Trp Gly Gln            100                 105                 110Gly Thr Thr Leu Thr Val Ser Ser         115                 120<210> 323 <211> 107 <212> PRT <213> Mus musculus <400> 323Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1               5                   10                  15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Asn Asn            20                  25                  30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile        35                  40                  45Lys Tyr Ala Ser His Ser Ile Ser Gly Ile Pro Ser Lys Phe Ser Gly    50                  55                  60Thr Gly Ser Gly Thr Asp Phe Thr Leu Ser Phe Asn Ser Val Glu Thr65                  70                  75                  80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu                85                  90                  95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys            100                 105 <210> 324 <211>   5 <212> PRT<213> Mus musculus <400> 324 Gly Asp Ser Ile Ala 1               5<210> 325 <211>   3 <212> PRT <213> Mus musculus <400> 325 Tyr Ser Gly 1<210> 326 <211>   4 <212> PRT <213> Mus musculus <400> 326Asp Tyr Phe Asp 1 <210> 327 <211>   7 <212> PRT <213> Mus musculus<400> 327 Arg Gln Asp Val Arg Lys Asn 1               5 <210> 328<211>   3 <212> PRT <213> Mus musculus <400> 328 Tyr Thr Ser 1 <210> 329<211>   6 <212> PRT <213> Mus musculus <400> 329 Tyr Asp Asn Leu Pro Phe1               5 <210> 330 <211> 114 <212> PRT <213> Mus musculus<400> 330Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln1               5                   10                  15Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Ala Ser Ala            20                  25                  30Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met        35                  40                  45Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys    50                  55                  60Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Gln Asn Gln Tyr Tyr Leu65                  70                  75                  80Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Val                85                  90                  95Thr Gly Asp Tyr Phe Asp Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val            100                 105                 110 Ser Ser<210> 331 <211> 107 <212> PRT <213> Mus musculus <400> 331Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Tyr Leu Gly1               5                   10                  15Gly Lys Val Thr Ile Thr Cys Lys Ala Arg Gln Asp Val Arg Lys Asn            20                  25                  30Ile Gly Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile        35                  40                  45Trp Tyr Thr Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly    50                  55                  60Ser Gly Ser Gly Arg Asp Tyr Ser Phe Asn Ile Asn Asn Leu Glu Pro65                  70                  75                  80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Pro Phe                85                  90                  95Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Arg            100                 105 <210> 332 <211>   5 <212> PRT<213> Mus musculus <400> 332 Gly Tyr Thr Phe Thr 1               5<210> 333 <211>   3 <212> PRT <213> Mus musculus <400> 333 Glu Thr Tyr 1<210> 334 <211>   4 <212> PRT <213> Mus musculus <400> 334Gly Tyr Pro Ala 1 <210> 335 <211>  12 <212> PRT <213> Mus musculus<400> 335 Ser Arg Thr Ile Leu His Ser Ser Gly Asn Thr Tyr1               5                   10 <210> 336 <211>   3 <212> PRT<213> Mus musculus <400> 336 Lys Val Ser 1 <210> 337 <211>   6 <212> PRT<213> Mus musculus <400> 337 Asp Ser His Val Pro Phe 1               5<210> 338 <211> 115 <212> PRT <213> Mus musculus <400> 338Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1               5                   10                  15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr            20                  25                  30Ser Met His Trp Val Lys Gln Ala Pro Gly Arg Gly Leu Lys Trp Met        35                  40                  45Gly Tyr Ile Asn Thr Glu Thr Tyr Glu Pro Thr Phe Gly Ala Asp Phe    50                  55                  60Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Ala Thr Thr Ala Tyr65                  70                  75                  80Leu Gln Ile Asn Ser Leu Lys Thr Glu Asp Thr Ala Thr Phe Phe Cys                85                  90                  95Gly Gly Gly Gly Tyr Pro Ala Tyr Trp Gly Gln Gly Thr Val Val Ile            100                 105                 110 Val Ser Ala        115 <210> 339 <211> 112 <212> PRT <213> Mus musculus <400> 339Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1               5                   10                  15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Arg Thr Ile Leu His Ser            20                  25                  30Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser        35                  40                  45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro    50                  55                  60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile65                  70                  75                  80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Asp                85                  90                  95Ser His Val Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys            100                 105                 110 <210> 340<211>   7 <212> PRT <213> Mus musculus <400> 340Gly Phe Asn Ile Lys Thr Thr 1               5 <210> 341 <211>   3<212> PRT <213> Mus musculus <400> 341 Ala Asp Asp 1 <210> 342 <211>   8<212> PRT <213> Mus musculus <400> 342 Phe Gly Tyr Val Ala Trp Phe Ala1               5 <210> 343 <211>   7 <212> PRT <213> Mus musculus<400> 343 Ser Gln Ser Val Asp Asn Tyr 1               5 <210> 344<211>   3 <212> PRT <213> Mus musculus <400> 344 Tyr Ala Ser 1 <210> 345<211>   6 <212> PRT <213> Mus musculus <400> 345 His Tyr Ser Ser Pro Tyr1               5 <210> 346 <211> 119 <212> PRT <213> Mus musculus<400> 346Glu Val Gln Leu Gln Gln Ser Val Ala Glu Leu Val Arg Pro Gly Ala1               5                   10                  15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Thr Thr            20                  25                  30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile        35                  40                  45Gly Arg Ile Asp Pro Ala Asp Asp Asn Thr Lys Tyr Ala Pro Lys Phe    50                  55                  60Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65                  70                  75                  80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ala Ala Ile Tyr Tyr Cys                85                  90                  95Val Arg Asp Phe Gly Tyr Val Ala Trp Phe Ala Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Phe Ser Ala         115 <210> 347 <211> 107 <212> PRT<213> Mus musculus <400> 347Asn Ile Val Met Thr Pro Thr Pro Lys Phe Leu Pro Val Ser Ser Gly1               5                   10                  15Asp Arg Val Thr Met Thr Cys Arg Ala Ser Gln Ser Val Asp Asn Tyr            20                  25                  30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile        35                  40                  45Tyr Tyr Ala Ser Asn Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly    50                  55                  60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Val65                  70                  75                  80Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Tyr Ser Ser Pro Tyr                85                  90                  95Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys            100                 105 <210> 348 <211>   7 <212> PRT<213> Mus musculus <400> 348 Gly Tyr Pro Phe Ser Glu Tyr1               5 <210> 349 <211>   3 <212> PRT <213> Mus musculus<400> 349 Glu Thr Gly 1 <210> 350 <211>   4 <212> PRT <213> Mus musculus<400> 350 Gly Tyr Pro Ala 1 <210> 351 <211>  12 <212> PRT<213> Mus musculus <400> 351Ser Arg Ser Ile Val His Ser Ser Gly Asn Thr Tyr1               5                   10 <210> 352 <211>   3 <212> PRT<213> Mus musculus <400> 352 Lys Val Ser 1 <210> 353 <211>   5 <212> PRT<213> Mus musculus <400> 353 Asp Ser His Val Pro 1               5<210> 354 <211> 115 <212> PRT <213> Mus musculus <400> 354Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1               5                   10                  15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Pro Phe Ser Glu Tyr            20                  25                  30Ser Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met        35                  40                  45Val Tyr Val Asn Thr Glu Thr Gly Gln Pro Ile Val Gly Asp Asp Phe    50                  55                  60Arg Gly Arg Phe Val Leu Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65                  70                  75                  80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys                85                  90                  95Gly Gly Gly Gly Tyr Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr            100                 105                 110 Val Ser Ala        115 <210> 355 <211> 112 <212> PRT <213> Mus musculus <400> 355Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1               5                   10                  15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Ile Val His Ser            20                  25                  30Ser Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser        35                  40                  45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro    50                  55                  60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile65                  70                  75                  80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Asp                85                  90                  95Ser His Val Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys            100                 105                 110 <210> 356<211>   7 <212> PRT <213> Mus musculus <400> 356Gly Phe Thr Phe Ser Ser Ser 1               5 <210> 357 <211>   3<212> PRT <213> Mus musculus <400> 357 Ala Thr Gly 1 <210> 358 <211>   8<212> PRT <213> Mus musculus <400> 358 Tyr Pro His Tyr Tyr Ala Met Asp1               5 <210> 359 <211>   7 <212> PRT <213> Mus musculus<400> 359 Ser Glu Asn Ile Phe Ser Asn 1               5 <210> 360<211>   3 <212> PRT <213> Mus musculus <400> 360 Ser Ala Thr 1 <210> 361<211>   6 <212> PRT <213> Mus musculus <400> 361 Phe Tyr Lys Ile Pro Phe1               5 <210> 362 <211> 121 <212> PRT <213> Mus musculus<400> 362Gln Gly Gln Met His Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ser1               5                   10                  15Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Ser Ser            20                  25                  30Phe Ile Ser Trp Leu Lys Gln Lys Pro Gly Gln Ser Leu Glu Trp Ile        35                  40                  45Ala Trp Ile Tyr Ala Ala Thr Gly Ser Thr Ser Tyr Asn Gln Lys Phe    50                  55                  60Thr Asn Lys Ala Gln Leu Thr Val Asp Thr Ser Ser Ser Ala Ala Tyr65                  70                  75                  80Met Gln Phe Ser Ser Leu Thr Thr Glu Asp Ser Ala Ile Tyr Tyr Cys                85                  90                  95Ala Arg His Ala Gly Tyr Pro His Tyr Tyr Ala Met Asp Tyr Trp Gly            100                 105                 110Gln Gly Thr Ser Val Thr Val Ser Ser         115                 120<210> 363 <211> 107 <212> PRT <213> Mus musculus <400> 363Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1               5                   10                  15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Phe Ser Asn            20                  25                  30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val        35                  40                  45Tyr Ser Ala Thr Asn Leu Gly Asp Gly Val Pro Ser Arg Phe Ser Gly    50                  55                  60Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro65                  70                  75                  80Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Phe Tyr Lys Ile Pro Phe                85                  90                  95Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys            100                 105 <210> 364 <211>   7 <212> PRT<213> Mus musculus <400> 364 Gly Phe Asn Ile Lys Asp Tyr1               5 <210> 365 <211>   3 <212> PRT <213> Mus musculus<400> 365 Glu Asp Gly 1 <210> 366 <211>   8 <212> PRT <213> Mus musculus<400> 366 His Gly Tyr Val Gly Trp Phe Ala 1               5 <210> 367<211>   8 <212> PRT <213> Mus musculus <400> 367Ala Ser Glu Asn Val Asp Thr Tyr 1               5 <210> 368 <211>   3<212> PRT <213> Mus musculus <400> 368 Gly Ala Ser 1 <210> 369 <211>   6<212> PRT <213> Mus musculus <400> 369 Ser Tyr Ser Tyr Pro Trp1               5 <210> 370 <211> 119 <212> PRT <213> Mus musculus<400> 370Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Pro Leu Lys Pro Gly Ala1               5                   10                  15Ser Val Lys Leu Thr Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp Tyr            20                  25                  30Tyr Ile His Trp Val Lys Gln Arg Ser Asp Gln Gly Leu Glu Trp Ile        35                  40                  45Gly Arg Ile Asp Pro Glu Asp Gly Glu Leu Ile Tyr Ala Pro Lys Phe    50                  55                  60Gln Asp Lys Ala Thr Ile Thr Val Asp Thr Ser Ser Asn Ile Ala Tyr65                  70                  75                  80Leu Gln Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95Ser Arg Asp His Gly Tyr Val Gly Trp Phe Ala Tyr Trp Gly Gln Gly            100                 105                 110Thr Leu Val Thr Val Ser Ala         115 <210> 371 <211> 107 <212> PRT<213> Mus musculus <400> 371Asn Val Val Met Thr Gln Ser Pro Lys Ser Met Ile Met Ser Val Gly1               5                   10                  15Gln Arg Val Thr Leu Asn Cys Lys Ala Ser Glu Asn Val Asp Thr Tyr            20                  25                  30Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile        35                  40                  45Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly    50                  55                  60Ser Arg Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala65                  70                  75                  80Glu Asp Leu Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Trp                85                  90                  95Thr Phe Gly Gly Gly Thr Lys Leu Glu Phe Arg            100                 105 <210> 372 <211> 219 <212> PRT<213> Artificial <220><223> an exemplary Pseudomonas exotoxin A variant 1(deimunized PE24 example)<400> 372Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser1               5                   10                  15Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His            20                  25                  30Ala Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr        35                  40                  45Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Ala Ala Arg    50                  55                  60Ser Gln Asp Leu Ala Ala Ile Trp Ala Gly Phe Tyr Ile Ala Gly Asp65                  70                  75                  80Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Ala                85                  90                  95Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Ala Ser            100                 105                 110Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu        115                 120                 125Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Ala    130                 135                 140Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr145                 150                 155                 160Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala                165                 170                 175Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser            180                 185                 190Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser        195                 200                 205Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys     210                 215<210> 373 <211> 219 <212> PRT <213> Artificial <220><223> an exemplary Pseudomonas exotoxin A variant 2(deimunized PE24 example)<400> 373Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser1               5                   10                  15Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His            20                  25                  30Ala Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr        35                  40                  45Ala Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg    50                  55                  60Ser Gln Asp Leu Arg Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp65                  70                  75                  80Pro Ala His Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg                85                  90                  95Gly Arg Ile Ala Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Ala Ser            100                 105                 110Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu        115                 120                 125Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg    130                 135                 140Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Glu Glu Thr145                 150                 155                 160Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala                165                 170                 175Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser            180                 185                 190Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser        195                 200                 205Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys     210                 215<210> 374 <211> 107 <212> PRT <213> Homo Sapiens <400> 374Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1               5                   10                  15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe            20                  25                  30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln        35                  40                  45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser    50                  55                  60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65                  70                  75                  80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                85                  90                  95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys            100                 105 <210> 375 <211> 105 <212> PRT<213> Homo Sapiens <400> 375Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu1               5                   10                  15Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe            20                  25                  30Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val        35                  40                  45Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys    50                  55                  60Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65                  70                  75                  80His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu                85                  90                  95Lys Thr Val Ala Pro Thr Glu Cys Ser             100                 105<210> 376 <211> 330 <212> PRT <213> Homo Sapiens <400> 376Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1               5                   10                  15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr            20                  25                  30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser        35                  40                  45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser    50                  55                  60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65                  70                  75                  80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                85                  90                  95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys            100                 105                 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro        115                 120                 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys    130                 135                 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145                 150                 155                 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                165                 170                 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu            180                 185                 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn        195                 200                 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly    210                 215                 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225                 230                 235                 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                245                 250                 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn            260                 265                 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe        275                 280                 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn    290                 295                 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305                 310                 315                 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                325                 330 <210> 377 <211> 330 <212> PRT<213> homo sapiens <400> 377Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1               5                   10                  15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr            20                  25                  30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser        35                  40                  45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser    50                  55                  60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65                  70                  75                  80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                85                  90                  95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys            100                 105                 110Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro        115                 120                 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys    130                 135                 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145                 150                 155                 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                165                 170                 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu            180                 185                 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn        195                 200                 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly    210                 215                 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225                 230                 235                 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                245                 250                 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn            260                 265                 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe        275                 280                 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn    290                 295                 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305                 310                 315                 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                325                 330 <210> 378 <211> 330 <212> PRT<213> homo sapiens <400> 378Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1               5                   10                  15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr            20                  25                  30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser        35                  40                  45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser    50                  55                  60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65                  70                  75                  80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                85                  90                  95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys            100                 105                 110Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro        115                 120                 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys    130                 135                 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145                 150                 155                 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                165                 170                 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu            180                 185                 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn        195                 200                 205Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly    210                 215                 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225                 230                 235                 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                245                 250                 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn            260                 265                 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe        275                 280                 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn    290                 295                 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305                 310                 315                 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                325                 330 <210> 379 <211> 327 <212> PRT<213> Homo Sapiens <400> 379Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1               5                   10                  15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr            20                  25                  30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser        35                  40                  45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser    50                  55                  60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65                  70                  75                  80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                85                  90                  95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro            100                 105                 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys        115                 120                 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val    130                 135                 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145                 150                 155                 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                165                 170                 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp            180                 185                 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu        195                 200                 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg    210                 215                 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225                 230                 235                 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                245                 250                 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys            260                 265                 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser        275                 280                 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser    290                 295                 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305                 310                 315                 320Leu Ser Leu Ser Leu Gly Lys                 325 <210> 380 <211> 280<212> PRT <213> homo sapiens <400> 380Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln Asn Ala Tyr Leu Pro1               5                   10                  15Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu Val Pro Val Cys Trp            20                  25                  30Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly Asn Val Val Leu Arg        35                  40                  45Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser Arg Tyr Trp Leu Asn    50                  55                  60Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr Ile Glu Asn Val Thr65                  70                  75                  80Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile Gln Ile Pro Gly Ile                85                  90                  95Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val Ile Lys Pro Ala Lys            100                 105                 110Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe Thr Ala Ala Phe Pro        115                 120                 125Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala Glu Thr Gln Thr Leu    130                 135                 140Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile Ser Thr Leu Ala Asn145                 150                 155                 160Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu Arg Asp Ser Gly Ala                165                 170                 175Thr Ile Arg Ile Gly Ile Tyr Ile Gly Ala Gly Ile Cys Ala Gly Leu            180                 185                 190Ala Leu Ala Leu Ile Phe Gly Ala Leu Ile Phe Lys Trp Tyr Ser His        195                 200                 205Ser Lys Glu Lys Ile Gln Asn Leu Ser Leu Ile Ser Leu Ala Asn Leu    210                 215                 220Pro Pro Ser Gly Leu Ala Asn Ala Val Ala Glu Gly Ile Arg Ser Glu225                 230                 235                 240Glu Asn Ile Tyr Thr Ile Glu Glu Asn Val Tyr Glu Val Glu Glu Pro                245                 250                 255Asn Glu Tyr Tyr Cys Tyr Val Ser Ser Arg Gln Gln Pro Ser Gln Pro            260                 265                 270Leu Gly Cys Arg Phe Ala Met Pro         275                 280<210> 381 <211> 181 <212> PRT <213> homo sapiens <400> 381Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln Asn Ala Tyr Leu Pro1               5                   10                  15Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu Val Pro Val Cys Trp            20                  25                  30Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly Asn Val Val Leu Arg        35                  40                  45Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser Arg Tyr Trp Leu Asn    50                  55                  60Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr Ile Glu Asn Val Thr65                  70                  75                  80Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile Gln Ile Pro Gly Ile                85                  90                  95Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val Ile Lys Pro Ala Lys            100                 105                 110Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe Thr Ala Ala Phe Pro        115                 120                 125Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala Glu Thr Gln Thr Leu    130                 135                 140Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile Ser Thr Leu Ala Asn145                 150                 155                 160Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu Arg Asp Ser Gly Ala                165                 170                 175Thr Ile Arg Ile Gly             180

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

The invention claimed is:
 1. A pharmaceutical composition comprising:(a) a T cell activating bispecific antigen-binding molecule, wherein theT cell activating bispecific antigen-binding molecule comprises a firstantigen-binding moiety that binds to CD3 and a second antigen-bindingmoiety that binds to Folate Receptor 1 (FoIR1), wherein the secondantigen-binding moiety comprises: (i) a complementarity determiningregion (CDR) heavy chain 1 (CDR-H1) comprising the amino acid sequenceof SEQ ID NO: 16, (ii) a CDR heavy chain 2 (CDR-H2) comprising the aminoacid sequence of SEQ ID NO: 17, (iii) a CDR heavy chain 3 (CDR-H3)comprising the amino acid sequence of SEQ ID NO: 18, (iv) a CDR lightchain 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 32,(v) a CDR light chain 2 (CDR-L2) comprising the amino acid sequence ofSEQ ID NO: 33, and (vi) a CDR light chain 3 (CDR-L3) comprising theamino acid sequence of SEQ ID NO: 34; and (b) a pharmaceuticallyacceptable carrier.
 2. The pharmaceutical composition of claim 1,wherein the T cell activating bispecific antigen-binding moleculefurther comprises a third antigen-binding moiety, wherein the thirdantigen-binding moiety binds to FoIR1.
 3. The pharmaceutical compositionof claim 2, wherein the third antigen-binding moiety comprises: (a) aCDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, (b) a CDR-H2comprising the amino acid sequence of SEQ ID NO: 17, (c) a CDR-H3comprising the amino acid sequence of SEQ ID NO: 18, (d) a CDR-L1comprising the amino acid sequence of SEQ ID NO: 32, (e) a CDR-L2comprising the amino acid sequence of SEQ ID NO: 33, and (f) a CDR-L3comprising the amino acid sequence of SEQ ID NO:
 34. 4. Thepharmaceutical composition of claim 3, wherein the third antigen-bindingmoiety is identical to the second antigen-binding moiety.
 5. Thepharmaceutical composition of claim 2, wherein at least one of thefirst, second, and third antigen-binding moiety is a Fab molecule. 6.The pharmaceutical composition of claim 1, wherein the secondantigen-binding moiety comprises a variable heavy chain comprising theamino acid sequence of SEQ ID NO: 15 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:
 31. 7. Thepharmaceutical composition of claim 1, further comprising a PD-1 axisbinding antagonist antibody.
 8. The pharmaceutical composition of claim7, wherein the PD-1 axis binding antagonist antibody is selected fromthe group consisting of a PD-1 binding antagonist antibody, a PD-L1binding antagonist antibody, and a PD-L2 binding antagonist antibody. 9.The pharmaceutical composition of claim 8, wherein the PD-1 axis bindingantagonist antibody is a PD-1 binding antagonist antibody.
 10. Thepharmaceutical composition of claim 8, wherein the PD-1 axis bindingantagonist antibody is a PD-L1 binding antagonist antibody.
 11. Thepharmaceutical composition of claim 8, wherein the PD-1 axis bindingantagonist antibody is a PD-L2 binding antagonist antibody.
 12. Thepharmaceutical composition of claim 1, wherein the first antigen-bindingmoiety and the second antigen-binding moiety are Fab molecules.
 13. Thepharmaceutical composition of claim 1, further comprising a T cellimmunoglobulin mucin 3 (TIM3) antagonist.
 14. A kit comprising: a T cellactivating bispecific antigen-binding molecule, wherein the T cellactivating bispecific antigen-binding molecule comprises a firstantigen-binding moiety that binds to CD3 and a second antigen-bindingmoiety that binds to FoIR1, wherein the second antigen-binding moietycomprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:16, (ii) a CDR-H2 comprising the amino acid sequence of SEQ D NO: 17,(iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, (iv)a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32, (v) aCDR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and (vi) aCDR-L3 comprising the amino acid sequence of SEQ ID NO:
 34. 15. The kitof claim 14, further comprising a package insert comprising instructionsfor using the T cell activating bispecific antigen-binding molecule witha PD-1 axis binding antagonist antibody to treat or delay progression ofa cancer in an individual.
 16. The kit of claim 15, further comprisingthe PD-1 axis binding antagonist antibody.
 17. The kit of claim 14,wherein the T cell activating bispecific antigen-binding moleculefurther comprises a third antigen-binding moiety, wherein the thirdantigen-binding moiety binds to FoIR1.
 18. The kit of claim 17, whereinthe third antigen-binding moiety comprises: (a) a CDR-H1 comprising theamino acid sequence of SEQ ID NO: 16, (b) a CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 17, (c) a CDR-H3 comprising the amino acidsequence of SEQ ID NO: 18, (d) a CDR-L1 comprising the amino acidsequence of SEQ ID NO: 32, (e) a CDR-L2 comprising the amino acidsequence of SEQ ID NO: 33, and (f) a CDR-L3 comprising the amino acidsequence of SEQ ID NO:
 34. 19. The kit of claim 18, wherein the thirdantigen-binding moiety is identical to the second antigen-bindingmoiety.
 20. The kit of claim 17, wherein at least one of the first,second, and third antigen-binding moiety is a Fab molecule.
 21. The kitof claim 14, wherein the second antigen-binding moiety comprises avariable heavy chain comprising the amino acid sequence of SEQ ID NO:15and a variable light chain comprising the amino acid sequence of SEQ IDNO:
 31. 22. The kit of claim 16, wherein the PD-1 axis bindingantagonist antibody is selected from the group consisting of a PD-1binding antagonist antibody, a PD-L1 binding antagonist antibody, and aPD-L2 binding antagonist antibody.
 23. The kit of claim 22, wherein thePD-1 axis binding antagonist antibody is a PD-1 binding antagonistantibody.
 24. The kit of claim 22, wherein the PD-1 axis bindingantagonist antibody is a PD-L1 binding antagonist antibody.
 25. The kitof claim 22, wherein the PD-1 axis binding antagonist antibody is aPD-L2 binding antagonist antibody.
 26. The kit of claim 15, wherein thecancer is selected from the group consisting of ovarian cancer, lungcancer, breast cancer, renal cancer, colorectal cancer, and endometrialcancer.
 27. The kit of claim 15, wherein the individual comprises lessthan about 15% PD-1 expressing tumor-infiltrating T cells.
 28. The kitof claim 14, wherein the first antigen-binding moiety and the secondantigen-binding moiety are Fab molecules.
 29. The kit of claim 14,further comprising a TIM3 antagonist.